One step methods, kits, and systems for measuring concentration of unbound bilirubin in biological fluids

Abstract

To provide improved techniques regarding identification and use of fluorescently labeled proteins that undergo a change in fluorescence index upon binding to bilirubin.SOLUTION: Disclosed herein are probes labeled at a cysteine or lysine residue. And probes labeled at both cysteine and lysine with two different fluorophores. These probes are useful for measurement of the level of unbound bilirubin in a fluid sample.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] This application claims the benefit of U.S. Provisional Application No. 62 / 894,553, filed August 30, 2019, which The entirety of which is expressly incorporated herein by reference.

[0002] This research was supported by SBIR Grant No. R44HD080412 from the National Institutes of Health. This invention was supported in part by the U.S. Government. Accordingly, the U.S. Government may have certain rights in this invention. Cut.

[0003] This application is based on FFASC077WOSEQLIST.TXT, created on August 24, 2020, which is 40KB in size. The information in the electronic sequence listing is incorporated by reference in its entirety. is expressly incorporated herein.

[0004] The present disclosure relates to the measurement of unconjugated bilirubin. [Background technology]

[0005] Bilirubin is a product of hemoglobin turnover and is poorly soluble in water, therefore , which is largely associated with albumin in plasma. However, a small amount of total plasma bilirubin is present in the aqueous phase. This unbound or free fraction can penetrate the blood-brain barrier and, at high levels, can be neurotoxic. Ahlfors CE, Wennberg RP, Ostrow JD and Tiribelli C, Unbound (free) bilirubin Bin: An improved paradigm for assessing neonatal jaundice. Clin Chem 55:1288-1299, 200 9]. Under normal conditions, total serum bilirubin is the regulated balance between bilirubin production and excretion. However, in newborns, the production-excretion balance is maintained at a low level by accumulation. Regulatory mechanisms may not be sufficiently mature to favor vasodilatory activity, and approximately 80% of newborns Jaundice often causes yellowing [Maisels MJ and McDonagh AF, Light for Neonatal Jaundice Radiotherapy, N Engl J Med 358: 920-928, 2008; Bhutani VK, Stark AR, Lazzeroni LC, P Holland R, Gourley GR, Kazmierczak S, et al. Pre-release testing for severe neonatal hyperbilirubinemia. Identifying infants who need phototherapy, J Pediatr 2013;162:477-82]. In some cases, this imbalance may be benign or actually beneficial, and most new Spontaneous resolution in live-born infants [Wennberg RP, Ahlfors CE, Bhutani VK, Johnson LH and and Shapiro SM, Understanding kernicterus: a challenge to improve the management of jaundiced newborns, Pe diatrics 117: 474-485, 2006; Gopinathan V, Miller NJ, Milner AD and Rice-Eva ns CA. Antioxidant activity of bilirubin and ascorbic acid in neonatal plasma. FEBS Lett 349: 197-200, 1994]. Unconjugated bilirubin levels can rise to neurotoxic levels. This can result in the more severe form of kernicterus, which can range from reversible hearing loss to, in rare cases, death. Disabilities ranging from severe to severe neurological sequelae occur [Ahlfors CE, Wennberg RP, Ostrow JD and d Tiribelli C Unbound (free) bilirubin: a revised paradigm for assessing neonatal jaundice Good, Clin Chem 55: 1288-1299, 2009].

[0006] Early intervention with phototherapy or exchange transfusion may reduce bilirubin-mediated neurotoxicity in newborns. [Maisels MJ and McDonagh AF, Phototherapy for neonatal jaundice, N Engl J Med 3 58: 920-928, 2008; Morris BH et al., Aggressive vs. Conservative Phototherapy for Very Low Birth Weight Infants. N Engl J Med 359:1885-1896, 2008; Kuzniewicz MW, Escobar GJ and Newman TB, I The impact of general bilirubin sieving on severe hyperbilirubinemia and phototherapy. Pediatrics 124: 1031-1039, 2009]. The intervention guidelines are based primarily on total bilirubin. It is value-dependent and takes into account gestational age and risk factors [Bhutani VK, Johnson L and Sivieri EM. Subsequent significant hyperbilirubinemia in healthy term and near-term neonates. Predictive ability of charge-time specific serum bilirubin for bilirubinemia. Pediatrics 103: 6-14. However, basic biochemical and increasing clinical evidence supports the use of total bilirubin. It is likely that unconjugated bilirubin correlates more accurately with bilirubin-mediated neurotoxicity than unconjugated bilirubin. Unbound (free) bilirubin: It is expected that neonatal jaundice will be Improving the paradigm for evaluating chemoattractants. Clin Chem 55: 1288-1299, 2009; Wennberg RP et al. New Intervention guidelines for neonatal hyperbilirubinemia: evidence-based quagmire, Curr Pharm Des 15: 2939-2945,2009; Ahlfors CE et al., Unconjugated bilirubin in a diverse neonatal population. Predicting abnormalities in automatic auditory brainstem responses in patients with rhesus monkeys, J Perinatol 29: 305309,2009; Oh W et al. Plasma total bilirubin and unconjugated bilirubin in very low birth weight infants and the risk of death or adverse events Influence of clinical status on associations with favorable neurodevelopmental outcomes. Acta Paediatr 99: 673-678, 201 0]. Therefore, to identify newborns at risk for bilirubin neurotoxicity, total bilirubin Unconjugated bilirubin should be preferable to lirubin [Ahlfors CE. Jaundice in the newborn]. Prediction of bilirubin neurotoxicity in children. Curr Opin Pediatr 22: 129-133, 2010; Watchko Bilirubin JF and Tiribelli C, Mechanisms and management approaches for induced neuropathy. N Engl J Med 2013 ; 369: 2021-30].

[0007] Aggressive phototherapy in premature infants should be designed to maintain total bilirubin below 5 mg / dL. Morris BH et al., Aggressive versus Conservative Phototherapy for Extremely Low Birth Weight Infants, NE ngl J Med 359: 1885-1896, 2008]. Morris et al. Outcomes (death and neurodevelopmental disability) in patients treated with ATP and those maintained at <8 mg / dL In a follow-up study, outcomes were measured by unbound bilirubin rather than total bilirubin. It has been shown that blood pressure in very low birth weight infants correlates well with blood pressure in very low birth weight infants [Oh W et al.]. Association of plasma total bilirubin and unconjugated bilirubin with mortality or adverse neurodevelopmental outcomes [Effect of clinical condition on the treatment, Acta Paediatr 99: 673-678, 2010]. Using total bilirubin to determine when to perform the test is important because the total bilirubin level is This may have been misleading because it was not conjugated with unbound bilirubin, the toxic fraction of bilirubin. This suggests the possibility of decoupling of total bilirubin and unbound bilirubin. g) can result from the presence of molecules that significantly interfere with bilirubin binding to albumin. For example, even if the total bilirubin is as low as 1 mg / dL, the total bilirubin due to interfering molecules may be Displacement of only 0.2% of the bilirubin results in 34 nM of unbound bilirubin. The unbound bilirubin level exceeds the level considered toxic to the infant, and Morris et al. Morris BH et al., Aggressive versus conservative phototherapy for extremely low birth weight infants, N Engl J Med 359: For premature infants such as those born in the United States (1885-1896, 2008), much lower unbound bilirubin levels are toxic. It was generally thought to indicate sexuality.

[0008] Several therapeutic and physiological processes can produce molecular variants of bilirubin. The natural form of bilirubin is the Z,Z isomer (Z,Z-bilirubin IXα). Upon exposure to light between 00 and 100 nm, three variants are produced; the photoisomers Z,E-bilirubin IXα and E,Z-bilirubin IXα, and the derivative Z-lumirubin IXα [Jana Ja Sproval et al., PLoS ONE DOI:10.1371, 2016; J. Jasheprova et al., Photodegradation of bilirubin The neurostimulatory effects of the product are reported in Scientific Reports (2018) 8:7444. Currently, the non-binding The only FDA-approved method for measuring combined bilirubin levels is HRP peroxidase. The bilirubin oxidation rate is measured by the arrows UB analyzer [HNakamura & Y , Lee, Microdetermination of unbound bilirubin in serum of jaundiced newborns: Peroxidase and bilirubin Enzymatic method using glucose oxidase, Clinica Chimica Acta, 79: 411-417, 1977]. The bilirubin photoproducts indicate that the decrease in bilirubin absorbance is due only to the Z,Z isomer. This may destroy the peroxidase assay for unbound bilirubin. The absorption spectra of the E,Z isomers overlap with the Z,Z absorbance. [Antony F. McDonagh h et al., Photoisomers: Confounding factors in clinical peroxidase assays of unconjugated bilirubin? Ped. iatrics 2009;123;67-76]. These and other photoisomers are much more abundant than the Z,Z isomer. Although soluble, it is much less toxic, so the peroxidase assessment was falsely elevated. In addition to bilirubin photoproducts, the liver conjugates bilirubin with glucuronic acid. It dissolves in bile and is excreted, where it eventually passes into the feces. Depending on the health of your liver, Some conjugated bilirubin may leak from the liver into the circulation. Nido modification increases solubility compared to unconjugated bilirubin, so conjugated bilirubin It is often present at concentrations of 2–50 μM with a reference range of 0–2 μM [Sanjiv Ha rpavat et al., Infants with persistent jaundice and normal newborns should have bilirubin measurements. Clinical Chemistry 61:2 330-334(2015)]. These μM concentrations are nM (0-100 nM) non-binding. Measurement of combined Z,Z bilirubin concentration, especially for easily distinguishing between bound and unbound bilirubin This may interfere with the non-specific peroxidase assay.

[0009] Many drugs and metabolites result in bilirubin being displaced from its bound state on albumin. Whether or not bilirubin can bind to albumin, thereby increasing total bilirubin levels Increased unbound lirubin concentration [Spear ML et al., 15-hour fat infusion with varying doses was Effect on bilirubin binding to albumin. JPEN J Parenter Enteral Nutr 9:144-147 , 1985 Amin SB. Bilirubin albumin binding affinity and unbound bilirubin in premature infants. Effect of free fatty acids on bin, JPEN J Parenteral Enteral Nutr 34: 414-420, 2010 Particularly important bilirubin-replacing metabolites are free fatty acids (FFA). Although present, it remains at low levels and does not have a significant effect on healthy, full-term newborns. However, under stressful conditions, such as those caused by sepsis, FFA concentrations may increase significantly. [Nogueira AC et al., Changes in plasma free fatty acid concentrations in sepsis patients may contribute to cardiac damage and [Associated with decreased heart rate variability, Shock 29: 342-348, 2008]. Preterm infants in the NICU In addition to stress, parenteral nutrition using oil emulsions such as Intralipid® can improve F It can cause an extremely large increase in FA concentration [Spear M et al., 15-hour fat injection Effect of acetaminophen on bilirubin binding to acetaminophen. JPEN J Parenter Enteral Nutr 9: 144-147, 1 985; Amin SB free fatty acids affect bilirubin-albumin binding affinity and unbound bilirubin in premature infants Effects on lirubin, JPEN J Parenteral Enteral Nutr 34: 414-420, 2010]FFA FFA binds to albumin with high affinity, similar to bilirubin. Unlike bilirubin, FFA Therefore, bilirubin is released only when a significant proportion of albumin binding sites are occupied by FFAs. The dose was varied to achieve significant substitution of 1000 ng / mL of a compound with multiple affinity binding sites [Spear ML et al. Effect of 5-hour fat infusion on albumin, JPEN J Parenter Nutr 9: 144-147, 1985; Amin SB, bilirubin-albumin binding affinity and unbound bilirubin in premature infants Effect of free fatty acids on the diet, JPEN J Parenter Nutr 34: 414-420, 2010] Intralipid( Newborns treated with GABA (registered trademark) showed a significant improvement in the quality of life of their offspring, suggesting that factors such as gestational age, enzyme activity, and fat accumulation may play a role. Therefore, the amount of FFA sufficient to replace bilirubin cannot be easily predicted. [Spear ML , et al., Effect of 15-hour fat infusion on bilirubin binding to albumin, JPEN J Paren ter Enteral Nutr 9: 144-147, 1985; Amin SB, Bilirubin-albuminuria in premature infants. Effect of free fatty acids on acetylcholine binding affinity and unbound bilirubin. JPEN J Parenter Ente ral Nutr 34: 414-420, 2010]. Monitor the unbound concentration of FFA (FFAu) during lipid infusion. When combined, elevated FFAu levels can significantly increase unbound bilirubin to dangerous levels. It was found that the effect of total bilirubin decoupling on the urinary tract infection was significantly improved [Hegyi T et al., Non-urinary tract infection treated with total bilirubin decoupling Bound free fatty acids are ineffective in phototherapy, Neonatology, 2013;104:184-187; Hegyi et al., Unbound Effect of soybean lipid infusion on free fatty acids and unconjugated bilirubin in rats, J Pediatr 2017; 184:45-50]. Furthermore, the unbound levels of these metabolites depend on many patient-specific factors. Therefore, unbound bilirubin is directly monitored during Intralipid® infusion. Only by doing so can infants at risk for bilirubin neurotoxicity be identified. This is due to the elevated plasma FFAs caused by increased Intralipid® concentrations. levels resulting in elevated unbound bilirubin concentrations without changing total bilirubin concentrations. This is especially true for bilirubin, as

[0010] Intracellular lipid-binding proteins (iLBPs) are a family of small, single-chain polypeptides. There are four recognized subfamilies: Subfamily I, which contains retinoic acid and proteins specific for vitamin A derivatives such as retinol. Subfamily II contains proteins with specificity for bile acids, eicosanoids, and heme. Subfamily III includes intestinal fatty acid binding proteins (FABPs). Subfamily IV includes all other types of lipids. Fatty acid binding proteins [Haunerland NH and Spener F, Fatty acid binding proteins, sinus from genetic manipulation] Prog Lipid Res 43: 328-349, 2004], which binds to bilirubin with low affinity. BP [Di Pietro SM and Santome JA. Two rat liver fatty acid binding protein isoforms. Isolation, characterization and binding properties of β-glucan. Biochim Biophys Acta 1478: 186 -200, 2000]. The entire family is characterized by a common tertiary fold. The ligand binding properties of the different subfamilies overlap considerably. Family I, [Richieri GV et al., Fatty acid binding proteins from different tissues are involved in the fatty acid phase Biochemistry 39: 7197-7204, 2000] and subf Family II both bind to fatty acids as well as their natural ligands. Single amino acid substitutions interalter the ligand binding properties of subfamily I and II proteins. [Jakoby MG et al., Ligand-protein electrostatic interactions can be and governing the specificity of fatty acid-binding proteins, Biochemistry 32: 872-878, 1993 The disclosure of each reference cited herein is incorporated by reference in its entirety. and is hereby expressly incorporated by reference. Summary of the Invention

[0011] Improved fluorophores, lower levels of interference from drugs and metabolites than other methods Interference allows for single-step Bf measurement using less than 5 μL of undiluted blood sample Near-infrared (NIR) Bf sensor with disposable cartridge and method of evaluation and calibration Described herein are compositions, kits, devices, systems, and methods for As described in, the probe may comprise iLBP labeled with a fluorophore. A probe for unbound bilirubin (Bf or UB) that becomes fluorescent when bilirubin binds. The index changes and can be used to measure the level of unbound bilirubin in a fluid. The fluorescence index can be measured, for example, by wavelength, intensity, polarization, lifetime, or fluorescence. The unconjugated bilirubin probes disclosed herein can be any measurable amount of The unbound bilirubin level is not significantly affected in the presence of other analytes present in the fluid being determined. The unbound bilirubin described herein does not bind or undergo a significant change in fluorescence. The probes are useful in the diagnosis and treatment of hyperbilirubinemia and in the treatment of bilirubin toxicity. It does not bind to bilirubin and can be used to assess the risk of It does not undergo a change in its fluorescence index and its fluorescence is present in the fluid in which the unconjugated bilirubin level is measured. Non-responsive probes that are generally not affected by other analytes present are also identified. The unconjugated bilirubin probe having a first fluorophore and a different fluorophore are then A non-responsive probe with a phore can generate a Bf sensor, which detects the presence of Bf Below, the change in the ratio of the fluorescence index from the first fluorophore to the second fluorophore is Together with a cartridge containing a Bf sensor and a dedicated fluorescence reader, is called the UBCheck assessment.

[0012] Some embodiments provided herein are directed to measuring free bilirubin in a sample. In some embodiments, the sensor is labeled with a first fluorophore. a bilirubin-responsive probe labeled with a second fluorophore and a non-responsive probe labeled with a second fluorophore. In some embodiments, the first and second fluorophores are excited at the same wavelength; The first and second fluorophores fluoresce at different wavelengths. In this case, the bilirubin-responsive probe contains the first intracellular lipid-binding protein (iLBP). , wherein the first iLBP has a peptide sequence comprising SEQ ID NO: 1, and Arginine replacing an accessible lysine (KR14 as shown in SEQ ID NO:2) Nin; a C-terminal double His tag linker (C 2XH11); N-terminal addition of MGI; and 62 or less amino acids containing a single cysteine. In some embodiments, the bilirubin-responsive probe comprises a bilirubin-responsive probe selected from the group consisting of: In some embodiments, the non-responsive probe comprises the sequence of any one of the probes described in The fragment contains a second iLBP, and the second iLBP contains a peptide sequence comprising SEQ ID NO: 1. and substitutions at positions 72, 73, 74, 126 and 131; Substitution to Cys at any one of positions 3, 74, 76, or 98; no more than three additional and a C-terminal double His having the sequence set forth in SEQ ID NO:3. In some embodiments, the non-responsive probe comprises a tag linker (C2XH11). In some embodiments, the first fluorophore comprises the sequence of any one of the probes described above. The fluorophore and the second fluorophore are different fluorophores. In this embodiment, the bilirubin-responsive probe is a single molecule having a first fluorophore attached thereto. In some embodiments, the non-responsive probe comprises a second fluorophore. In some embodiments, the first fluorophore comprises a single cysteine ​​to which the second fluorophore is attached. The first and second fluorophores are excited at the same or nearly the same wavelength. In this form, the first fluorophore is attached to a cysteine ​​substitution in the LICOR700DX male. imide or LICOR800CW maleimide. The bilirubin-responsive probe is constructed to bind to the unconjugated IX-α(Z,Z) isomer of bilirubin. In some embodiments, the bilirubin-responsive probe is a probe capable of binding bilirubin (4 mg). In some embodiments, the ubiquitin-containing benzoate is configured to minimally bind to benzoyl benzoate (less than 1 / dl). The bilirubin-responsive probes are the Z,E or E,Z photoisomers of bilirubin, lumirubin, and fatty acids. configured so as not to bind to any other naturally occurring blood components and / or neonatal drugs In some embodiments, the neonatal drug is not spironolactone. In some embodiments, the non-responsive probe is unbound IX- of bilirubin or bound bilirubin. In some embodiments, the non-reactive α(Z,Z) isomer is configured not to bind to the α(Z,Z) isomer. The probes are the Z,E or E,Z photoisomers of bilirubin, lumirubin, fatty acids, any It is configured so as not to bind to other naturally occurring blood components and / or neonatal drugs. In some embodiments, the first fluorophore is LICOR700DX maleimide. The second fluorophore was attached to a cysteine ​​substitution of LICOR800CW maleimide. and the first fluorophore is LICOR800CW maleimide, the second fluorophore is In some embodiments, the fluorophore is LICOR700DX maleimide. wherein the first fluorophore or the second fluorophore is attached to a cysteine ​​substitution; cysteine ​​substitutions are 22, 24, 25, 26, 27, 29, 30 of SEQ ID NO: 1 In some embodiments, the first The emission intensity of the first fluorophore or the second fluorophore is determined by the bilirubin and hemoglobin In some embodiments, the absorbance of the blood component is not affected by the absorbance of the blood component selected from the group consisting of: The bilirubin-responsive probe or the non-responsive probe further comprises at least one linker. This includes:

[0013] Some embodiments provided herein may include any of the sensors described herein. In some embodiments, the composition comprises free bilirubin (Bf) sensor. In some embodiments, the sensor binds to bilirubin and comprises a first fluorophore. The first intracellular lipid-binding protein (iLBP) is labeled with and does not bind to bilirubin. and a second iLBP labeled with a second fluorophore, wherein the second fluorophore is a second iLBP. 1 is not bound to iLBP, and the first fluorophore and the second fluorophore are in the same wavelength range. The first and second fluorophores are excited at different wavelengths, and the emission wavelengths of the first and second fluorophores are different. The fluorophore of 2 does not change its emission in the presence of bilirubin. In this embodiment, the first fluorophore is LICOR700DX maleimide and the second fluorophore is LICOR700DX maleimide. The first fluorophore is LICOR800CW maleimide or the second fluorophore is LI The first fluorophore is COR800CW maleimide and the second fluorophore is LICOR700DX maleimide. In some embodiments, the change in the ratio of the fluorescence indices is and is used to determine the concentration of unbound bilirubin. In the case of the first fluorophore or the second fluorophore, the emission intensity of the bilirubin and It is not affected by the absorbance of blood components selected from hemoglobin.

[0014] Some embodiments provided herein include the sensors described herein or The present invention relates to a solid substrate comprising the composition described herein. The Rubin-responsive probes and / or non-responsive probes are attached to a solid substrate. In some embodiments, the solid substrate is Ni-polystyrene, Ni-latex, or Ni- In some embodiments, Ni-polystyrene, Ni-latex, In some embodiments, the bile acid, bismuth, or Ni-agarose beads contain iron. The bin-responsive or non-responsive probes may contain the substitution 7R as set forth in SEQ ID NO:3. 16R 20R 29R 37R 46R 50R 88R 92R 94R 100R 125 R includes 129R and / or 130R (KR14). The lirubin-responsive probe and / or the non-responsive probe comprise a tag, and the solid substrate comprises a tag. In some embodiments, the tag is a His-tag, biotin, Flag-epi Tope, c-myc epitope, HA-tag, glutathione-S-transferase (GST ), maltose-binding protein (MBP), chitin-binding domain (CBD), thioredoxin scin, β-galactosidase, VSV glycoprotein, calmodulin-binding protein, These include one or more of the following: a polystyrene (PS) hydrophobic tag, or a metal affinity tag. In some embodiments, the tag is a polyhistidine tag and the solid substrate comprises an immobilized metal chelate. In some embodiments, the first fluorophore is a bilirubin-responsive probe. In some embodiments, the second fluorophore is attached to a cysteine ​​residue on the is attached to a cysteine ​​residue on the non-responsive probe.

[0015] Some embodiments provided herein use bilirubin to determine Kd and Rm. In some embodiments, the method comprises calibrating a bin sensor. Mixing any one of the sensors with an aqueous sample of known concentration of bilirubin Bt; and measuring the light intensity and comparing it with the measured fluorescence by applying the following equation (1): and determining positive parameters:

[0016]

number

[0017] where R is the measured fluorescence ratio ((I λ1 / I λ2 ), I λ1 is the background from the sample. is the fluorescence intensity from the first fluorophore at wavelength λ1 minus I λ2 Is it a sample? is the fluorescence intensity from the second fluorophore at wavelength λ2 minus the background. , Ro is the ratio in the absence of bilirubin, BT is the total bilirubin concentration, and PT is the response is the probe concentration, and r is the bilirubin probe fluorophore in the absence of the second fluorophore. Rophore I λ2 / I λ1 is the ratio, and Kd is the equilibrium dissociation constant of the bilirubin probe. Rm is the ratio R extrapolated to infinite BT.

[0018] Some embodiments provided herein provide a method for determining the concentration of free bilirubin [Bf] in a sample. In some embodiments, the method comprises measuring the baseline fluorescence of a sample. measuring and applying the sample to any one of the sensors described herein measuring sample fluorescence; and subtracting the baseline fluorescence from the sample fluorescence to obtain the measured fluorescence. and determining the concentration of [Bf] from the measured fluorescence. In some embodiments, the steps of measuring a baseline and / or subtracting a baseline are The step of:

[0019]

number

[0020] In some embodiments, equation (1) is used to calibrate the sensor, and equation (2) is f], where R is the measured fluorescence ratio ((I λ1 / I λ2 ) Ri, I λ1 is the fluorescence intensity from the first fluorophore at wavelength λ1, and I λ2 is the wavelength λ is the fluorescence intensity from the second fluorophore at 2 and Ro is the ratio in the absence of bilirubin and r is the I of the probe in the absence of the second fluorophore. λ2 / I λ1 is the ratio, Kd is the dissociation constant, Rm is the minimum R value at ∞Bf, and Rm is the bilirubin R at saturation.

[0021] In some embodiments, the sample contains one or more carrier macromolecules for bilirubin. In some embodiments, the one or more carrier macromolecules are albumin, lipids, or the like. In some embodiments, the soluble ... In some embodiments, the sensor is attached to a solid support. The concentration of ATP is determined using a disposable microfluidic device, which can optionally be diluted and In some embodiments, the sample may be human, animal, or In some embodiments, the sample is derived from whole blood, plasma, serum, urine, CSF, In some embodiments, the sample is an oil emulsion. In some embodiments, the sample is from a patient receiving an intravenous infusion of from patients receiving drugs that displace bilirubin from albumin, and / or Or such patients may displace bilirubin from albumin from the injected oil emulsion. In some embodiments, the sample may be subjected to phototherapy, transfection, or other blood, or from patients undergoing other treatments that lower bilirubin levels. In some embodiments, Ro is obtained by photobleaching the sample, thereby obtaining a zero level Bell measurements are obtained.

[0022] Some embodiments provided herein relate to cartridges. In some embodiments, the cartridge is configured to measure bilirubin in a sample. In an embodiment, the cartridge includes a substrate and a sample receiving device configured to couple to the substrate. a lens with a sample port for detecting bilirubin, a bilirubin-responsive probe, a non-responsive probe, and and a substrate having an anti-hemoglobin peptide immobilized thereon. In one embodiment, the substrate is treated with UV light having a wavelength in the range of about 145 nm to about 225 nm, thereby As a result, the photo-treated polystyrene polymer chains are linked to the polymer chains of the substrate. In embodiments, the substrate is a polystyrene substrate. Some include a material having a dark color configured to reduce the reflection intensity of excitation light at 100 nm. In embodiments, the lens is an acrylic lens. In some embodiments, the lens is an O2 In some embodiments, the lens is bonded to a polystyrene substrate. This creates a groove with a depth of approximately 0.1 mm or less, sealing the cartridge. In some embodiments, the sample is whole blood, plasma, serum, urine, CSF, saliva, gastric juice, interstitial fluid, or In some embodiments, the sample is an undiluted sample. In some embodiments, the cartridge is configured to measure bilirubin at equilibrium. In some embodiments, the cartridge is calibrated with a traceable bilirubin standard. In some embodiments, bilirubin standards are used to calibrate the probe. Commercially available bilirubin, the probe is used to calibrate the calibration complex, the calibration complex is used to calibrate the cartridge.

[0023] Some embodiments provided herein relate to kits. In this regard, the kit includes one or more collection devices for collecting samples from a patient, Any one of the sensors as mounted on the any one of the compositions as described herein, and below medically determinable levels and and / or one or more reference standards containing known concentrations of unconjugated bilirubin. In some embodiments, one or more reference standards are optional. [Brief explanation of the drawings]

[0024] [Figure 1]1 depicts an embodiment of the emission spectra of a LICOR700DX maleimide-labeled bilirubin-sensitive probe and a LICOR800CW maleimide-labeled non-responsive probe mixed in a fluorometer, showing the intensity at zero Bt and titration of the mixture with increasing Bf, which shows quenching of emission at 710 nm, no change at 805 nm, and a corresponding decrease in the 710 / 805 ratio. [Figure 2] 2 shows an embodiment of free probe calibration data with fits and resulting parameters. Figure 2 shows calibration data for an embodiment of a free probe with a concentration of 1.2 nM, fitting Equation 1 yielding the parameters Kd, Rm and Qs = Rm / Ro. [Figure 3] An embodiment of the calibration of a cartridge lot using a calibrated bilirubin-human serum albumin (HSA) complex that produces a fixed Bf value is shown. The R values ​​measured at each Bf were fitted to Equation 3 to determine the quality of fit, weighted by Kd (nM), Rm, and χ. [Figure 4] 1 shows an embodiment of the effect of dilution on Bf levels in the presence of oleic acid, a strong substitute. [Figure 5] The concentration of unbound bilirubin (Bf) as a function of hemoglobin (Hb) dilution is shown. [Figure 6] An embodiment of the effect of a neonatal intensive care unit (NICU) drug that is a potent displacer of bilirubin from albumin is shown. [Figure 7] An embodiment of Bf assessment is shown, demonstrating less sensitivity to conjugated bilirubin (cBR) than the Arrows peroxidase method. [Figure 8] Figures 8A and 8B show an embodiment that reduces Bf partitioning to triglycerides in the absence of lipolysis (Figure 8A), and activates lipolysis in the presence of heparin, increasing intralipid concentrations to generate unbound FFAs that increase Bf by displacing bilirubin from albumin (Figure 8B). [Figure 9]9A and 9B show embodiments of data demonstrating that the Bf assay detects only the Z,Z isomer of bilirubin, while Arrows is sensitive to Z,Z and all photoisomers. [Figure 10] 10A-10D schematically represent multi-views of an embodiment of a polystyrene disposable sample cartridge substrate. [Figure 11] 11A-11D schematically depict multi-views of an embodiment of a lens configured to couple to the polystyrene disposable sample cartridge substrate shown in FIGS. 10A-10D. [Figure 12] FIG. 12 shows a disposable cartridge with the Bf sensor spot and sample port clearly marked. DETAILED DESCRIPTION OF THE INVENTION

[0025] In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. The symbol "like" typically identifies similar components unless the context dictates otherwise. The illustrative embodiments set forth in the following description, drawings, and claims are not intended to be limiting. It is not intended to depart from the spirit or scope of the subject matter presented herein. The embodiments may be utilized and other changes may be made. The aspects of the present disclosure shown in the drawings can be arranged, substituted, combined, and / or configured in a wide variety of different configurations. It is understood that various modifications may be made, isolated, and designed, all of which are expressly contemplated herein. It will be easily understood.

[0026] U.S. Patent Nos. 5,470,714, 6,444,432, 7,601,510, 9,134,317, and 9,52 Nos. 9,003 and 9,817,004, which are incorporated herein by reference, are incorporated by reference in their entirety. These protocols are expressly incorporated herein by reference in their entirety. The proteins from the intracellular lipid-binding protein (iLBP) family are either native or As mentioned above, this family is composed of either a mutated form or a fatty acid-binding Fatty acid-binding protein A family proteins (FABPs) [Banaszak et al., and retinoid transport protein Adv Protein Chem 45:89-151, 1994; Bernlohr DA et al. Intracellular lipid-binding proteins and their genes. Annu Rev Nutr 17:277303, 1997. LBP is an intracellular protein with a molecular weight of approximately 15 kDa, and the wild-type protein is 1 or two FFAs as well as other metabolites.

[0027] Each patent and publication disclosed and described herein is incorporated by reference in its entirety. Any disclosure specifically incorporated herein by reference in any way is hereby incorporated by reference in its entirety. Free or attached proteins that do not bind or respond to fluorophores and bilirubin The present invention is based on the bilirubin-sensitive iLBP, which is composed of a bilirubin-sensitive iLBP having a second fluorophore attached thereto. This paper describes an unbound bilirubin (UB) also called free bilirubin (Bf) ratio sensor. The disclosure provides, for example, near-infrared (NIR) Bf sensors with improved fluorophores, other methods Interference from lower levels of drug and metabolites, less than 5 μL of undiluted blood Disposable cartridges enabling single-step Bf determination in samples and comparative evaluation Improvements over prior patents and publications, including positive methods, are described.

[0028] The bilirubin-sensitive NIR fluorescently labeled iLBP mutants described herein have improved The bilirubin specificity and sensitivity of the antibody were improved. No. 9,529,003 is fluorophore-labeled primarily at lysines, terminal amino groups, or cysteines. As disclosed in U.S. Patent No. 9,134,317, Labeling with TECH was performed at multiple sites (wild-type FABP (sequence shown in SEQ ID NO: 1) FABP proteins (including proteins having the FABP gene) or mutant FABP proteins (e.g., those shown in Tables 1 and 2) The mutant protein having the sequence shown does not have a cysteine ​​residue) By eliminating the need for ion channels, sensitivity and specificity for detecting ligand binding to iLBP are improved. Fluorophores at most sites change their fluorescence upon ligand binding. Labeling at most sites results in a high fluorescence signal-to-noise ratio upon ligand binding. In addition to the terminal amino group, iLBP has 14 surface-accessible lysines. One improvement of the present disclosure is to provide a method for labeling fluorophores, all of which can be labeled with amino-reactive fluorophores. New availability of two fluorophores: LICOR700DX and LICOR800CW LICOR700DX Maleimide Mido was not available or known at the time of previous disclosure, and LICOR800CW Maleimides were not studied in U.S. Pat. No. 9,529,003. Bilirubin-sensitive iLBP is a specific antibody labeled with LICOR700DX-maleimide. It has a single cysteine ​​mutation. It shows no change in fluorescence in the presence of bilirubin, and is LICOR8. We also developed iLBP mutants labeled with single cysteine ​​mutations using 00CW-maleimide. It is being issued.

[0029] The probes disclosed herein are compatible with over 50 of the most prescribed drugs for neonates (e.g., For example, those listed in Table 4), conjugated bilirubin, photoisomers of bilirubin, premature or Intravenous lipid emulsions used to provide parenteral nutrition to other at-risk infants The antibody may be insensitive to α- and free fatty acids.

[0030] The present disclosure also relates to a device for determining the concentration of unbound bilirubin in a sample. In some embodiments, the device includes a blood sample microchannel (e.g., 1-50 μL, In some embodiments, the NIR fluorescence is measured as a dried spot in the center of the sample (with a volume of 5 μL). It contains a disposable plastic cartridge containing a Bf sensor. The fluorescence from the cartridge is by inserting the cartridge into a fluorescence reader specially developed for this purpose. The blood sample can be measured after application. This configuration of the reader allows the blood level of unbound bilirubin to be increased in a single step by several steps. The configuration may allow measurements to be made using microliter volumes of sample.

[0031] Drying the Bf sensor on the cartridge reduces the bilirubin-sensitive probe's fluorescence. This is useful for characterizing the sensor and for characterizing bilirubin. The ratio of the fluorescence from the bilirubin-sensitive probe divided by the fluorescence from the bilirubin-insensitive probe New analytical methods for the calculation of conjugated bilirubin concentrations are needed.

[0032] Thus, some embodiments provided herein provide a high degree of unconjugated bilirubin. Specific fluorescently labeled proteins (or iLBP mutant proteins) and bilirubin Method for identifying fluorescently labeled iLBP mutant proteins that are unresponsive to steroids Some embodiments are directed to the bilirubin-responsive iLBP mutant protein and Different near-infrared (NIR) fluorophores on non-responsive iLBP mutant proteins When combined in a Bf sensor, the presence of Bf is This results in a change in the ratio of the refractive indexes of the R fluorophores. Range from aqueous solutions to complex biological samples including human fluids (blood, CSF, urine, interstitial fluid) To fabricate a fluorescence ratio sensor for determining the unbound bilirubin concentration in a sample, two In some embodiments, the method includes using the fluorescently labeled protein of The probes are made using the methods of U.S. Pat. Nos. 7,601,510 and 9,529,003. Fabricating a ratiometric Bf sensor by the use of a second fluorescently labeled non-responsive protein One or both of the fluorescent proteins may be free in solution. or one or both of which are solid substrates such as polydextran or polystyrene. In some embodiments, the polymer may be attached to a matrix or resin. , the method calibrates the sensor to determine the sensor's calibration constants using Eq. (1). Some embodiments involve attaching the probe to a solid substrate. The characteristics of the ligated probes are that they allow the positive detection of unbound bilirubin levels in such devices. Such features can be tested to assess the accuracy and precision of the determination. These may include the effects of separation, albumin buffering, or bilirubin binding to the polymer, but the polymer In some embodiments, the Bf sensor specific Common metabolites, drugs, bilirubin photoisomers, conjugated bilirubin, or concentrations less than 1 nM Including unbound bilirubin or other analyte contributions equivalent to some medically relevant level. This can be refined by testing against a panel of potential interfering substances, including: In this embodiment, the method further comprises determining specificity for unbound bilirubin in a human blood sample. To confirm the accuracy, a defined non-binding assay was performed in human plasma, serum, or whole blood with added bilirubin. In some embodiments, the method further comprises testing the quantification of bilirubin by measuring the bilirubin. Further comprising calculating the unbound bilirubin concentration as set forth in equation (2).

[0033] Some embodiments include SEQ ID NO: 1, which includes one or more amino acid substitutions and a fluorophore. For probes based on iLBP, such as the lipid binding protein corresponding to ID NO: 1 (See examples in Tables 1 and 2.) In some embodiments, fluorophores The carboxyl group is attached to a cysteine ​​residue of iLBP, which has only a single reactive cysteine. In some embodiments, bilirubin-sensitive iLBP binds to the IXα-Z,Z isomer of bilirubin. It binds to the photoisomers Z,E, E,Z and lumirubin, conjugated bilirubin, or fatty acids. The non-reactive iLBPs also contained only a single reactive cysteine, and these samples does not bind to or respond to any of the

[0034] In some embodiments, the bilirubin-sensitive probe comprises an N-terminal MGI substitution, a C-terminal The double HIS tag substitution C2XH11 (SEQ ID NO: 3), and SEQ ID NO :1, 14, 18, 23, 28, 24, 25, 26, 27, 29, 30, 33, 38, 5 From 4, 60, 73, 74, 76, 97, 98, 106, 115, 117, or 132 A lipid-binding fragment of SEQ ID NO:1 having one or more amino acid substitutions at selected positions Corresponding to the fusion protein.

[0035] In some embodiments, the fluorophore for the bilirubin-responsive probe is 22, 24, 25, 26, 27, 29, 30, 33, 54, and 73 of SEQ ID NO:1 , 74, 76, 97 or 98 cysteine ​​substitutions.

[0036] In some embodiments, the probe detects a mutation at position 29 to cysteine. Except for 7R16R20R29R37R46R50R88R92 of SEQ ID NO:1 R94R100R125R129R and / or 130R (KR14-SEQ ID N O:2) is substituted with arginine.

[0037] In some embodiments, the probe has the sequence RGAASHHHHHHSHRATPNTS PHHHHHHH (SEQ ID NO:3) at the C-terminus of SEQ ID NO:1 Includes car (C2XH11).

[0038] In some embodiments, the polynucleotide template is a cleavable or non-cleavable affinity In some embodiments, the template polymerase encodes an iLBP mutant protein having a tag. The oligonucleotide template encodes an iLBP mutant protein with a polyhistidine affinity tag. The solid substrate comprises an immobilized metal chelate.

[0039] In some embodiments, bilirubin-responsive and non-responsive iLBP mutant proteins The protein is labeled with a single fluorophore at a pH below 8. In some embodiments, the fluorophore can react with a cysteine ​​side chain. The thiol specific amine is excited at a wavelength of approximately 660 nm and emits at wavelengths of approximately 700 nm and 819 nm. heterofluorophores, e.g., LICOR700DX-maleimide and LICOR80 0CW maleimide.

[0040] In some embodiments, the second fluorophore is a non-responsive iLBP mutant protein. This is provided by the addition of a fluorophore to a non-responsive probe such as a protein. BP also contains a single fluorophore that reacts preferentially with cysteine ​​side chains at pH below 8. In some embodiments, the fluorophore is LICOR800CW-Malay In some embodiments, the non-responsive i LBP fluorescence compared with bilirubin-responsive (bilirubin-binding) iLBP mutein probe and had zero or significantly reduced response in its fluorescence index upon exposure to bilirubin. do.

[0041] In some embodiments, the non-responsive probes comprise one or more amino acid substitutions and a full iLB-like lipid-binding protein corresponding to SEQ ID NO: 1, including a urophore P (see examples in Table 2). In some embodiments, the fluorophore It is conjugated to a cysteine ​​residue in iLBP, which has only a single reactive cysteine.

[0042] In some embodiments, the non-responsive probes are 14, 18, 20, 23, 27, 2 9, 33, 54, 72, 73, 74, 76, 98, 100, 117, 126, or 13 1. A lipid-binding molecule of SEQ ID NO:1 having one or more amino acid substitutions at positions selected from Corresponding to the fusion protein.

[0043] In some embodiments, the fluorophore for the non-responsive probe is SEQ I Cysteine ​​at position 27, 31, 33, 54, 73, 74, 76, or 98 of D NO:1 Attached to the substitution.

[0044] Some embodiments provided herein involve the use of iL labeled with a first fluorophore. BP mutant protein and a separate unbound iLBP that does not bind bilirubin. In some embodiments, the present invention relates to compositions comprising a first fluorophore and a second fluorophore. The fluorophore and the second fluorophore can be excited at the same wavelength, and the first fluorophore The emission wavelengths of the first fluorophore and the second fluorophore are different. The second fluorophore binds bilirubin to the bilirubin-unresponsive iLBP mutant protein. Unaffected (does not change its release) and / or non-responsive iLB in response to binding P does not bind to bilirubin. In some embodiments, the first fluorophore is LIC The first fluorophore is OR700DX maleimide, and the second fluorophore is LICOR800CW maleimide. It's Mido.

[0045] In some embodiments, the change in the ratio of fluorescence indices is measured at two different wavelengths, and this ratio is Used to determine unbound bilirubin concentration.

[0046] In some embodiments, the index is a fluorophore attached to the iLBP mutein. The luminescence intensity of the α-glucan is the intensity of the luminescence of bilirubin and hemoglobin, as described herein. It is not significantly affected by the light absorption (above 600 nm) of blood components such as thiamin. In some embodiments, the systems and methods described herein are hemolysis independent. This causes significant interference from hemoglobin and heme in the sample. The methods, systems, and compositions provided herein overcome the obstacles associated with hemolysis. In some embodiments, the peptide is added to a solid substrate to which the Bf probe is attached. , and this peptide eliminates or significantly reduces interference from hemolysis.

[0047] In some embodiments, the first fluorophore is attached to a cysteine ​​and The second fluorophore is 700DX-maleimide, and the second fluorophore is also cis-fluorophore on a different iLBP. The ligated cation is LICOR800CW-maleimide.

[0048] In other embodiments, the first fluorophore is LICOR800CW-maleimide. , conjugated to a bilirubin-sensitive mutein, and the second fluorophore is LICOR700DX It is a cleavable imide and is attached to the non-responsive mutein.

[0049] In other embodiments described herein, the second, different fluorophore is a bilirubin. Bilirubin-responsive iLBP mutant protein is bound to a protein that does not bind to bilirubin. A first fluorophore bound to a protein and a probe bound to a second fluorophore are The bound probe binds to the bilirubin-unresponsive iLBP mutant protein and therefore Therefore, the mixture of responsive and non-responsive probes produces two different markers in response to bilirubin. The second fluorophore produces a Bf sensor that changes the ratio of the fluorescence indices measured at the different wavelengths. The fluorophore is longer or shorter than the first (responsive iLBP mutant protein) fluorophore. Both fluorophores should have a common excitation wavelength. For example, in some embodiments, the first (protein-bound) fluorophore is a LIC OR700DX maleimide, and the second example is LICOR800CW maleimide, and / or Biotium CF800 maleimide conjugated to bilirubin-unresponsive proteins One or both of the fluorescently labeled proteins may be in solution. It may be free in the polymer or embedded in another polymer or solid substrate. This arrangement ensures that the excitation maximum of the second fluorophore is aligned with the excitation maximum of the first fluorophore. The fluorophores are aligned so that the emission intensities of both fluorophores are similar even when the lengths are different. This has the desirable advantage that the concentration of the first fluorophore can be adjusted. This type of assay uses a second, different fluorophore that is not bound to the same protein as Ratio probes are typically used when both fluorophores are located on the same protein. Eliminates the problem of energy transfer quenching of one fluorophore by the other .

[0050] In some embodiments, the second fluorophore is attached to an acceptor protein. In some embodiments, the probe is designed so that one of these positions is a cysteine. SEQ ID NO:1 except with substitutions 7R16R20R29R37R 46R50R88R92R94R100R125R129R and 130R (KR14- SEQ ID NO:2).

[0051] In some embodiments, the probe has the sequence RGAASHHHHHHSHRATPNTS C-terminal linker C2XH11 consisting of PHHHHHHH (SEQ ID NO: 3) Includes.

[0052] Embodiments provided herein include those in which the fluorophore is a cysteine ​​residue (e.g., LIC OR700DX Maleimide, LICOR800CW Maleimide, LICOR, IRDye6 80LT Maleimide, Alexafluor680 Maleimide or BiotiumCF800 Maleimide ) is attached to the probe.

[0053] In some embodiments, any of the above probes may be used for attachment to a solid support. Two or more tags may be attached to the C- or N-terminus of the probe in combination with one or more linkers for It may include

[0054] In some embodiments, the probe comprises two His tags and two linkers. is attached to a solid support using

[0055] Embodiments provided herein are directed to compositions comprising such probes. .

[0056] Some embodiments provided herein provide a bilirubin-sensitive probe free in solution. (e.g., LICOR700DX-maleimide) and bilirubin-unresponsive probes (e.g., In some embodiments, the methyl group is a maleimide. The bilirubin-sensitive and bilirubin-insensitive probes are attached to a solid substrate in solution. do.

[0057] The embodiments provided herein include any of the above-described probes attached to a solid substrate. In some embodiments, the solid substrate optionally comprises iron. Polystyrene or latex beads, Ni-polystyrene beads. The probes selected for attachment to the substrate may be either alone or N- and C-terminally modified. , including but not limited to, substitution of the linker and surface lysine (KR14), The two probes with different emission wavelengths may contain any of the following modifications in combination: They may be immobilized on the same or different solid substrates, which may be nanoparticles or beads.

[0058] In some embodiments, the probes are tagged for attachment to a solid substrate. In some embodiments, the tag is a His tag, biotin, a Flag epitope, a c-myc epitope, or the like. pitope, HA tag, glutathione-S-transferase (GST), maltose bond MBP, chitin-binding domain (CBD), thioredoxin, β-galactosidase tosidase, VSV-glycoprotein, calmodulin-binding protein, polystyrene (P S) hydrophobic tag, or metal affinity tag.

[0059] In some embodiments, the probe has a tag and the solid substrate comprises a receptor for the tag. In some embodiments, the tag may or may not have an additional linker. The polyhistidine tag and the solid substrate comprises an immobilized metal chelate.

[0060] Other embodiments include dextran, polystyrene, latex, agarose beads, or including polymers such as Ni-NTA polystyrene beads (optionally containing iron), These include, but are not limited to, bilirubin probes attached to solid substrates that are nanoparticles. These nanoparticle substrates can be further immobilized on macroscopic surfaces. An example of the use of such surfaces is in the grooves of disposable microfluidic devices (single-use sample cartridges). Immobilization on a surface includes, but is not limited to, Examples of designed bilirubin probes include combinations of each from Tables 1 and 2. The double His tag and linker may include, but are not limited to, polystyrene, Including but not limited to Ni, Co, or Cu on latex or agarose beads This allows for chelation with a variety of metal ligands on a variety of polymer resins. These beads with attached lobes can be used freely in solution and are suitable for bilirubin reactions. Responsive and non-responsive probes can be attached to the same or different beads, and both configurations For the synthesis, two different fluorophores eliminate energy transfer, thereby In some embodiments, the separation is sufficient to obtain a specific response to Rubin binding. In some embodiments, the substrate is a microfluidic device or a multiwell plate. In this embodiment, the substrate is attached to the surface of the cartridge by a detection device as described herein. Included in the chair.

[0061] The embodiments provided herein include a method for preparing a cysteine-containing antibody having a single cysteine ​​labeled with a fluorescent dye. In some embodiments, the present invention relates to LBP muteins. Any surface lysine or any other system that has fluorescent labeling activity under lysine-specific labeling conditions. The amino acid may be replaced with another amino acid, including, for example, alanine or arginine. In some embodiments utilizing an iLBP mutein template corresponding to ID NO: 1 However, lysine at position 27 is highly reactive and typically does not bind unless a label is directed at that position. Typically, it can be mutated to alanine.

[0062] In some embodiments, the bilirubin is albumin, a lipid binding protein, a lipid vesicle, or Bilirubin forms a complex with a carrier polymer such as cyclodextrin. The complex with buffers the concentration of unbound bilirubin, clamping the level of unbound bilirubin. In some embodiments, the carrier polymer is albumin. In terms of morphology, albumin is more soluble in bilirubin than, for example, bovine serum albumin. Human serum albumin (HSA) has a high affinity for ATP and therefore has been shown to be a key In embodiments, albumin buffer may be more preferred for bilirubin.

[0063] The embodiments provided herein provide a bilirubin preparation that, in an aqueous medium, increases bilirubin concentration (BT). The lirubin sample and the sensor were mixed, and the ratio of the fluorescence intensity R of the sensor was measured at each concentration, and the ratio was calculated using Equation (1). Calibration parameters (Kd, Rm, and Ro) were calculated from the measured fluorescence by fitting ), where R is The measured fluorescence ratio (I λ1 / I λ2 ), where I λ1 is the first fluorophore at wavelength λ1. is the fluorescence intensity from the fluorophore, and I λ2 is the fluorescence from the second fluorophore at wavelength λ is the intensity, λ1 and I λ2 Both are background subtracted, and Ro is bilirubin. BT is the total bilirubin concentration, and PT is the concentration at the sensor. where r is the fluorescence I of the bilirubin-sensitive probe in the absence of the second fluorophore λ2 / I λ1 is the ratio, Kd is the equilibrium dissociation constant of the sensor, and Rm is the value extrapolated to infinite BT The ratio R is the ratio of the number of ions in the saturation region. Equation (1) is not Equation (5) of U.S. Pat. No. 9,529,003 when Rm > 0. and is used in some embodiments.

[0064] The embodiments provided herein include optionally measuring the fluorescence of the sample and mixing with a sample and measuring fluorescence, and optionally, measuring the sample fluorescence in the presence of a sensor. Intensity: The fluorescence intensity of the sample in the absence of the sensor (background or blank) and calculating R from the background-subtracted sensor intensities; and determining the concentration of [Bf] from formula (2). This invention relates to a method for measuring bilirubin [Bf] concentration.

[0065] In some embodiments, Equation 2 is used to calibrate the sensor and / or measure Bf. where R is the measured fluorescence ratio ((I λ1 / I λ2 ) and I λ1 is the wavelength λ is the fluorescence intensity from the first fluorophore at 1, and I λ2 is the second fluorophore at wavelength λ2. is the fluorescence intensity from the fluorophore and the intensity of both minus the sample blank, and Ro is the fluorescence intensity from the fluorophore. is the ratio in the absence of lirubin, and r is the I of the sensor in the absence of the second fluorophore. λ2 / I λ1 is the ratio, Kd is the dissociation constant, Rm is the minimum R value at ∞Bf, and Rm is , R at bilirubin saturation of the probe.

[0066] In some embodiments, the sensor comprises a bilirubin-responsive iLBP and one fluorophore. It consists of two fluorophores in combination, and the second fluorophore is in solution. bound to free polymers or proteins free in solution, and does not bind to bilirubin In some embodiments, the sensor binds or responds to bilirubin and is attached to a solid substrate. It consists of a protein with one fluorophore and the second fluorophore is also A virilization that is attached to a solid substrate but is separated from the protein bearing the first fluorophore. It attaches to another protein that does not bind or respond to the bin.

[0067] In some embodiments, the sample contains albumin, lipid binding proteins, lipid vesicles, or synaptosomes. Carrier polymers for bilirubin such as chlorodextrin.

[0068] In some embodiments, the bilirubin-responsive probes and the non-responsive probes are disposable. The device is then attached to the channel of a microfluidic channel, enabling measurement of Bf in undiluted blood samples. To perform Noh.

[0069] In some embodiments, the sample is from a human, animal, or plant. In this case, the sample may be from whole blood, plasma, serum, urine, CSF, saliva, gastric juice, interstitial fluid, or lymph. In some embodiments, the sample is subjected to an intravenous infusion of an oil emulsion. In some embodiments, the sample is from a patient, such as from a disease or stress It is derived from patients who are able to produce molecules that displace bilirubin from albumin. In this embodiment, the sample is obtained from a patient being treated with a drug capable of displacing bilirubin from albumin. In some embodiments, the sample is derived from a subject who has undergone phototherapy, a blood transfusion, or bilirubin levels. The data were from patients receiving other treatments that reduce blood cholesterol.

[0070] The embodiments provided herein include one or more collection devices for collecting samples from a patient. device, one or more sensors as above, or one or more sensors in a suitable carrier and optionally a reference standard containing a known concentration of unconjugated bilirubin. This applies to kits that are

[0071] The embodiments provided herein relate to probes defined in either Table 1 or 2. Let's call it an elephant.

[0072] For purposes of this disclosure, bilirubin is the Z,Z isomer of unconjugated bilirubin IXα [M cDonagh AF et al. Photoisomers: a confounding factor in clinical peroxidase assays of unconjugated bilirubin. Pediatrics 123: 67-76, 2009] Unbound bilirubin is generally present in plasma as albumin. Unbound Z, ZIXα bilirubin differs from bilirubin found bound to α- It is an aqueous monomer of

[0073] For purposes of this disclosure, the term "lipid" shall be construed as having its ordinary and accustomed meaning, Mostly soluble in organic solvents, but with some level of solubility in the aqueous phase (unbound fraction) Thus, the term "lipid-binding protein" refers to a compound that binds lipids to the lipids in the present specification. The term "protein" includes any protein that is capable of binding to lipids, as defined above.

[0074] Levels of unbound molecules, such as bilirubin, lipids including fatty acids, hormones, and metabolites When measured in appropriate human or animal body fluids, the assay provides diagnostic information for health and disease. The unbound (herein referred to as "aqueous phase" or "free") concentrations of such molecules can be Determining the concentration (also called the level) provides important information about physiological homeostasis. It is becoming increasingly clear that many metabolites are hydrophobic molecules with low water solubility. , which has an unbound concentration much lower than the "total" concentration, and the majority of the "total" is protein or In biological fluids, the concentration of unbound molecules is low under normal physiological conditions. This adjustment is often adjusted to maintain a relatively constant unbound concentration at 1000 kJ / s. This occurs through interactions with carrier proteins such as albumin. The majority of the molecules are generally bound to albumin or other carriers. However, a small number of molecules They may dissociate (and re-associate) from albumin into the aqueous phase, and these may be unbound. He is a child.

[0075] For purposes of this disclosure, in some embodiments, a "bilirubin sensor" is a sensor that detects cysteine ​​residues. Two iLBPs labeled with two different fluorophores at the fluorophore group, where the first i LBP undergoes a change in fluorescence index when bound to bilirubin, and the second iLBP binds to bilirubin ( In some embodiments, the fluorescence of the non-responsive probe does not change significantly in the presence of the non-responsive probe. Bilirubin sensors can also be free in solution, attached to different molecules or polymers, and The second fluorophore does not change fluorescence in the presence of lirubin. The additional fluorescence provided by the iLBP may include fluorescently labeled iLBP at the cysteine ​​residue. In this case, binding to bilirubin changes the fluorescence of only one of the fluorophores. When the probe is unbound, the ratio of the fluorescence indices at the two wavelengths is different. can be used to specifically determine the aqueous concentration of rubin, which is otherwise For example, due to its poor solubility in aqueous solutions and the presence of other metabolic products, especially free fatty acids. The change in the ratio of the fluorescence response is difficult to accurately measure the intracellular concentration of unbound bilirubin. This is particularly important for accurate determination of the extracellular concentration of unbound bilirubin and improves the accuracy and precision of the determination. It is important to improve.

[0076] U.S. Patent Nos. 7,601,510, 9,134,317 and 9,529,003, [Huber AH et al., Fatty Acids and Their Related Compounds] Mixtures of different fluorescent probes and different unbound free fatty acids in equilibrium with albumin Their use in separating substances, Biochemistry, 45:14263-14274, 2006] and [Huber AH and Kleinfeld AM. Unbound free fatty acid profiles and unbound palmitoyltransferases in human plasma. The unexpected absence of oleic acid [J. Lipid Res. 58:578-585, 2017] may be a key factor in determining unbound analytes. We describe a method for the high-throughput generation of highly specific probes that allow for the determination of U.S. Patent Nos. 7,601,510, 9,134,317 and 9,529,003 disclose bilirubin-specific The embodiments provided herein are in accordance with U.S. Pat. 0, 9,134,317, and 9,529,003, and related to improvements in bilirubin technology. Embodiments of the present disclosure improve the accuracy and precision for the determination of unconjugated bilirubin levels, This technology allows for use in different measurement formats. U.S. Patent No. 7,601,51 Nos. 0, 9,134,317, and 9,529,003 [Huber et al., Determination of Unbound Bilirubin Concentration] [Clin Chem 58: 869-876, 2012] Robe primarily uses probes in aqueous suspension (cuvette-based fluorescence measurements) , using diluted plasma or serum samples. The embodiments disclosed herein involve two probes: Using iLBP mutant proteins labeled with near-infrared fluorophores The first iLBP emitted fluorescence at 700 nm. It is labeled with LICOR700DX-maleimide, which emits fluorescence, and its fluorescence binds to Z,Z bilirubin. The second iLBP fluoresces at 819 nm and does not respond to bilirubin. Labeled with LICOR800CW-maleimide. Labeled with LICOR700DX-maleimide. Both LICOR800CW-maleimide can be excited at 660 nm. Some embodiments provided relate to the quenching of long wavelength probes by bilirubin. Additionally, some embodiments include a method for attaching a bilirubin probe to a solid surface; Unbound bilirubin in microfluidic devices and disposable sample cartridges The present invention relates to a method of using such a composition to measure bottles.

[0077] Bilirubin sensors are used to determine the unbound bilirubin level in a blood sample, Fatty acids are the most abundant metabolic products in the blood, with properties similar to bilirubin. competes with bilirubin for binding to albumin and has an unbound concentration similar to that of unbound bilirubin. Bilirubin-sensitive probes generally have a high affinity for fatty acids. Therefore, the iLBP mutant protein is developed. The first step in discovering bilirubin probes from the probes was to identify probes that did not respond significantly to fatty acids. Over 300,000 fatty acids were identified using up to 11 of the most abundant fatty acids to identify lobes. The aim is to screen such probes. Generally, mutations without fluorescent labels are detected. The mutant protein does not induce a measurable signal upon analyte binding. Systematic high-throughput screening of quality is not possible. The finding that 2<0.1 would have resulted in over 10,000 such fatty acid non-responders ("non-responders"). This quantitative benchmark is the basis for the identification of these proteins for fatty acids. The affinity of the probe is generally at least 10-fold less than the ADIFAB2 reference probe. By screening these non-responsive probes with bilirubin, new Further mutagenesis of the identified template protein led to the development of new mutein probes. Potential bilirubin probes and / or templates used for library generation The resulting library was screened for responses to fatty acids and bilirubin. It is cleaned and is most responsive to bilirubin and to fatty acids. The probe identified as the least responsive was identified as the bilirubin probe or or used as templates for further rounds of mutagenesis and screening. This is achieved by measuring the bilirubin and non-responsiveness of the blood as described herein (e.g., Tables 1 and 2). The results showed that the bilirubin sensor showed a significant response to fatty acids. There is no answer.

[0078] Usefulness includes significant response to bilirubin and zero for low response to fatty acids Bilirubin probes identified by these methods as having desirable properties will be further characterized. The probes that do not significantly respond to FFAs by binding to FFAs are bilirubin. In some embodiments, binding to FFA is 10 times less than binding to bililamine. This is due to the probe's bilirubin binding affinity and fluorescence. Calibration to determine characteristics, as well as bilirubin to identify potential competition with fatty acids and monitor unbound bilirubin levels in aqueous solutions containing human serum albumin. This involves the production of non-responsive probes in which fatty acids bind to the probe but do not produce a change in fluorescence. In this case, fatty acids in the blood sample compete with bilirubin for binding to the probe. may combine, thereby resulting in an inaccurate determination of unbound bilirubin levels. Competition with fatty acids occurs when the fluorescence response of bilirubin probe + bilirubin decreases with the addition of fatty acids. It is evaluated by determining whether it changes.

[0079] Provides accurate bilirubin concentration and detectable in solutions containing bilirubin and albumin Bilirubin proteases found by the methods described herein that do not exhibit significant fatty acid competition Individual neonatal and adult donors are selected for further testing in human blood samples. Plasma samples from donors, as well as pooled samples from commercial sources, were analyzed for bilirubin protease activity. The probe has essentially unknown levels of non-bilirubin analytes commonly present in human blood samples. Determine whether the test provides accurate serum or plasma unbound bilirubin concentrations in samples Healthy adults have low bilirubin levels and a bilirubin:HSA molar ratio of The Bf concentration in these blood samples is less than 0.1, and therefore approaches zero (<1 nM). Samples were spiked with bilirubin and albumin concentrations were measured to detect values ​​below 1 nM. Obtain a clear bilirubin:albumin ratio that results in a Bf level above the limit of deviation (LOD). The concentration of unbound bilirubin in various samples was measured using a Bf sensor, and the results were compared with those obtained using peroxidase. Oxidase assay [Jacobsen J and Wennberg, RP]. Quantitation of unbound bilirubin in neonatal serum. The quantitative determination of unbound bilirubin in milk was compared with that in Clin Chem 20: 783, 1974. An FDA-cleared study on the Arrows UB-2 analyzer [Nakamura H and Lee Y, Icteric Neoplasia Microdetermination of unconjugated bilirubin in infant serum: Peroxidase and glucose oxidase. The procedure was carried out using an enzymatic method using enzymes [Clinica Chimica Acta, 79 (1977) 411-417]. Plasma unbound bilirubin concentrations measured by Bf sensor and peroxidase assay Equivalence with unbound bilirubin indicates that blood components other than unbound bilirubin have no detectable effect on probe performance. Make sure that it does not.

[0080] U.S. Patent Nos. 7,601,510, 9,134,317 and 9,529,003 are tamper-evident claims that should be omitted. This requires a necessary and time-consuming step prior to characterization of bilirubin binding to proteins; Only the probe itself is characterized. This is not only to avoid characterization of the protein. However, the properties of the probe are often not predictable from the ligand-protein binding properties. For example, different proteins may have very similar binding affinities, but The fluorescence response of these derivative probes can vary.

[0081] Most previously described bilirubin probes primarily target the lysine residues of SEQ ID NO:1. 27 was labeled with only acrylodan (U.S. Patent Nos. 7,601,510, 9,134,317, and and 9,529,003, and Huber et al., Fluorescence Sensing for Quantitating Unbound Bilirubin Concentration. (Clin Chem 58:869-876, 2012). Additional bilirubin probes were added to the two Different fluorophores, i.e., acrylodan at lysine 27 of SEQ ID NO:1 and and N-terminal adduct cysteine ​​labeled with Texas Red maleimide in two versions. One without KR14 and the other without KR14 ("KR14" is 7R16R2 0R29R37R46R50R88R92R94R100R125R129R and 13 14 surface lysine to arginine mutations, including 0R. is an abbreviation for acrylonitrile, having substitutions as shown in SEQ ID NO:2, These probes have good affinity for bilirubin. and response, and is not significantly affected by non-bilirubin metabolites in human blood samples. However, the bilirubin concentration in the specimen was high, and the pathology of severe neonatal hyperbilirubinemia [Bhuta ni VK and Johnson L, Jaundiced newborns in the emergency department: preventing kernicterus, Clin Ped Emer g Med 9:149-159, 2008], and the presence of hemoglobin in blood samples It is possible that the Dan-only probe adversely affects the bilirubin-mediated inner filter effect. Acrylodan and longer wavelength fluorophores (e.g., Texas Red) Dual-labeled probes are due to energy transfer between acrylodan and the secondary fluorophore. This significantly reduces the acrylodan fluorescence intensity, making such probes unlikely to be used at clinically relevant concentrations. It is not possible to obtain an exact value for Bf in the range of degrees.

[0082] To overcome these drawbacks, the embodiments provided herein provide a method for treating unconjugated bilirubin. The present invention relates to a sensor and method for determining the level of bilirubin-quenched full-antibody. The fluorophore labels a single cysteine ​​side chain, and the position of this side chain determines the fluorescence upon bilirubin binding. A mutein library was identified that was found to be important for optimizing the changes. Fluorophores that are quenched by bilirubin label different cysteine ​​side chains, and the side chains The position has been found to be important for optimizing the fluorescence change upon bilirubin binding. A bilirubin probe has also been described. Bilirubin-quenching fluorophores that absorb and emit at long wavelengths where bilirubin quenching should not occur. The present disclosure also describes very long wavelength fluorophores, including those that extend into the infrared. Regarding the fluorescence quenching of fluorophores by bilirubin, their long wavelength absorbance and fluorescence For this purpose, such fluorophores are used to measure bilirubin or hemoglobin absorbance, or blood It is unaffected by the absorbance of substantially any other chromophores potentially present in the liquid sample.

[0083] Further embodiments described herein include a single full-length antibody on a bilirubin-binding protein. It is produced using a fluorophore, and its fluorescence decreases when it binds to bilirubin. Binds to proteins that are unresponsive and / or do not bind to bilirubin (non-responsive probes) Method for producing a bilirubin ratio sensor using a second, different fluorophore Such sensors measure the change in the ratio of fluorescence indices measured at two different wavelengths. , which responds to bilirubin binding to the protein portion of the probe. This type of ratio sensor uses a fluorophore, where both fluorophores are in the same molecule, such as a protein. Energy transfer quenching between fluorophores, which is typically observed when they are located on polymers, Such quenching significantly reduces signal intensity, thereby eliminating the problem of non- This avoidance of energy transfer reduces the accuracy and precision of the measurement of the bound bilirubin concentration. , is achieved by not attaching both fluorophores to the same probe molecule.

[0084] Also, beads that can be attached to solid substrates such as polystyrene or latex beads. Lirubin probe, and table for use in disposable microfluidic devices Beads that can be immobilized on a surface are also described. are attached to bilirubin-responsive iLBP and bilirubin-unresponsive iLBP, respectively. In some embodiments, two full-length fluorescein ... Both probes can be applied to the same or different fixed sites so that the oligophores remain well separated. The beads can be attached to a disposable plastic microfiber substrate. Sensor beads can be immobilized on a fluidic device in an aqueous buffer solution (slurry). The complex mixture was dispensed into the grooves of the microfluidic device in a volume of less than 2 μl and allowed to dry. The undiluted blood forms a circular spot of approximately 2 mm in diameter and is deposited at the bottom of the groove. The sample is applied to the entrance of the channel and flows rapidly across the dried bilirubin sensor spot. The blood sample-containing device is then reconstituted with the bilirubin-responsive probe and the non-responsive probe. The fluorescence from the sensor is then measured by a fluorescence reader, from which the Bf Calculate the concentration.

[0085] The bilirubin sensors described herein can also be calibrated and used to determine the Bf concentration in a single A method for determining the amount of ATP in an undiluted blood sample of approximately 5 μl is also described. These small blood samples are delivered to a disposable plastic tube containing a dry bilirubin ratio sensor. The bilirubin sensor containing the microfluidic cartridge is preferably are used for a single measurement and the calibration parameters (Kd, Rm, Ro) are manufactured identically. Calibration must be determined within a given "lot" of cartridges. This is done by titrating the ridge with aqueous samples containing increasing levels of distinct Bf. The stick binds unbound bilirubin and has a large surface-to-volume ratio within the microfluidic channel. Therefore, the calibration Bf samples must be highly buffered by complexing with albumin. The Bf concentration of each complex was measured by cuvette fluorescence using a free calibrated sensor in aqueous solution. The free sensor is bilirubin, which is determined by measuring each complex by photometry. The concentration of these unbound bilirubin solutions was determined by titration with an aqueous solution of The response of the free probe (R value vs. Bf concentration) is used to determine the free sensor Determine the calibration (Kd, Rm, Ro) (Equation 1). Mix each BR:HSA complex sample with Using the calibrated free sensor, calculate each complex by cuvette fluorometry using Equation 2. These calibrated complexes are suitable for blood samples at various temperatures, p Under the defined conditions of H and solution composition, the equilibrium dissociation constant (Kd) and minimum fluorescence ratio (Rm) Used to determine the binding parameters of the disposable cartridges, including the initial ratio (Ro) and The binding isotherm is the change in fluorescence of the bilirubin sensor in response to increasing bilirubin concentrations. The titration data is performed in aqueous buffer by measuring the titration of each bilirubin ("titration data"). The set of fluorescence responses at concentrations as a function of Bf concentration, specific spectral properties, and Kd It is fitted with an appropriate equation ("calibration equation (3)") that accurately describes the fluorescence response.

[0086]

number

[0087] Free bilirubin concentration ([Bf]) is the concentration at which [Bf] is buffered by the albumin-bound equilibrium. and therefore determined in samples that are not significantly perturbed by the presence of the bilirubin probe. Equation (2) is used to determine [Bf] for ratio sensors where Rm > 0.

[0088] Some embodiments provided herein involve the use of a method for determining the concentration of unbound analyte. More particularly, some embodiments relate to the development of fluorescent protein molecules that can be used in: 1) Produced by the methods of U.S. Patent Nos. 7,601,510, 9,134,317, and 9,529,003 These methods are expressly incorporated herein by reference with respect to the identification of selected bilirubin probes. 2) for clinical medicine and basic science; or 3) the use of such probes to determine the concentration of unbound bilirubin in different fluids. Regarding the examples of these probes for determination.

[0089] The bilirubin probe contains one or more fluorescent probes that exhibit a change in fluorescence index upon binding to bilirubin. iLBP proteins that are "labeled" by the covalent attachment of molecules (fluorophores) In some embodiments, the probe is a single sequence with a covalently attached fluorophore. Contains stains.

[0090] In some embodiments, two different fluorophores are used, One of the probes responds to bilirubin binding, and when bilirubin binds to the probe, a fluorescence index is observed. The second fluorophore does not respond to bilirubin, but rather to iLBP, which indicates changes in bilirubin. The second probe is used to label the iLBP that binds bilirubin or that does not bind. This provides a reference point so that the difference in the ratio of fluorescence at different wavelengths can be observed. may not react with bilirubin binding or may react in a different manner than the first fluorophore. In some embodiments, the second fluorophore may react with the first fluorophore. A chemical dye that can be used as a second fluorophore has an emission point at a wavelength different from that of the first fluorophore. Examples of materials include LI-COR800CW maleimide, Cy7 maleimide, and Cy7.5 maleimide. These include Benzylimide, VivoTag-S750-M, and Alexa Fluor 750. In some embodiments, the second fluorophore is LI-COR800. CW maleimide.

[0091] In some embodiments, one fluorophore is attached to a cysteine, and this fluorophore The fluorophore is responsive to bilirubin binding, i.e., bilirubin binds to the fluorescently labeled iLBP mutein. Two different fluorophores were used, demonstrating a change in fluorescence index upon binding to fluorophores. The second fluorophore is not chemically conjugated to the bilirubin-binding iLBP mutein, but rather to the iLBP mutein. The second fluorophore is not sensitive to bilirubin binding to the BP mutein. Provides a reference point as the difference in the ratio of fluorescence at two different wavelengths upon binding is observed. In one embodiment, the second fluorophore is at a longer wavelength than the first fluorophore. Examples of chemical dyes that can be used as the first fluorophore include LI -COR700DX Maleimide, BiotumCF680-M, CF680R-M, Lumiprobe Cy5, Cy7, Perkin Elmer Vivo Tag 645-M, Vivo Tag 680XL-M, Atto Tek A tto680, Atto700, DyomicsDY677, or DY689. Examples of chemical dyes that can be used as second fluorophores include, but are not limited to, LI In some embodiments, the hydroxyl group may be selected from the group consisting of hydroxyl groups, ... One fluorophore is LI-COR700DX maleimide and the second fluorophore is is LI-COR800CW maleimide.

[0092] The iLBP mutant proteins were designed so that they bind to solid supports with high affinity. These may be "tagged" with biotin, Flag-epitope or c-myc epitope. HA-tag, glutathione-S-transferase (GST), maltose Binding protein (MBP), chitin-binding domain (CBD), thioredoxin, β-galactosidase ctosidase, VSV-glycoprotein, calmodulin-binding protein, polystyrene ( (PS) tagging with a hydrophobic tag or a metal affinity tag such as a 6XHis tag. The specific association of an affinity tag with a solid support material can be achieved by, but is not limited to, including but not limited to multi-well plates and microfluidic devices This facilitates the measurement of unbound bilirubin in a flat surface configuration. Due to the attachment of the probes, the probes can be concentrated into a limited, effectively two-dimensional area. allows measurement of unbound bilirubin in a thin layer of sample solution flowing across the probe This is limited to a valid two-dimensional area. This effectively enables front-face fluorescence measurement, which reduces absorbance due to the ion exchange reaction and facilitates measurements in whole blood. The affinity tag may be attached to the NH2-terminus or COOH-terminus, for example, as shown in Tables 1 and 2. In some embodiments, the 6X fusion may be performed at either end or both ends simultaneously. The histidine tag can be used to bind iL without significantly altering the bilirubin-binding properties of the protein. BP mutant proteins at either the NH2-terminus or COOH-terminus, or at both termini. In some embodiments, the fusion peptide is a CO It consists of two separate histidine tracts at the OH-terminus. In the present invention, the probe is mounted on a solid support, including but not limited to Ni-polystyrene beads. It becomes fixed.

[0093] In some embodiments, the bilirubin sensor immobilized on the solid support is excited at the same wavelength. Two probes labeled with different fluorophores that emit at two different wavelengths are used. One of the two probes is responsive to bilirubin binding. When bilirubin binds to proteins, it shows a change in fluorescence index. The second probe, labeled with a fluorophore, either does not respond to bilirubin binding or does not bind bilirubin. The fluorophore of the second probe changes differently in response to lirubin than the first probe. The difference in the ratio of fluorescence at two different wavelengths is observed upon bilirubin binding. In some embodiments, the first bilirubin-sensitive protein is LI -COR700DX is a maleimide-labeled, second bilirubin-insensitive protein. -COR800CW labeled with maleimide.

[0094] Some embodiments provided herein are shown in FIGS. and 12, which relate to disposable sample cartridges containing Bf sensors. After adding, the cartridge is placed in a fluorescence reader to measure the Bf concentration of the sample. In some embodiments, the cartridge is In some embodiments, the lens is constructed from a polystyrene base and an acrylic lens. So, the sensor is bilirubin sensitive iLBP from Table 1, LICOR700DX from Table 2. - composed of maleimide and non-responsive iLBP, LICOR800CW-maleimide, The two probes are attached to Ni-NTA polystyrene beads either on separate beads or on the same bead. The antibody is coupled to a fluorophore bead (e.g., Dynal 1 μm NTA beads). The conditions are listed in Table 4 to help reduce interference from hemoglobin and / or hemolysates. These peptides are added to Ni-NTA polystyrene beads with bound probes. The probe and peptide-labeled beads are suspended in an aqueous buffer to form a slurry, which Dispense the Bf sensor beads onto a polystyrene substrate as droplets with a volume of 250–2000 nL. The spots are dried, and then channels containing the dried sensor spots are formed. In some embodiments, the polystyrene substrate is encapsulated by an acrylic lens. Before application, the slurry is treated with UV radiation. In some embodiments, the UV radiation is about 185 nm. UV irradiation at wavelengths of 1000 nm immobilizes polystyrene bead sensors and clearly depicts them on the polystyrene substrate. It is superior to 254 nm illumination in forming a well-defined circular sensor spot. In some embodiments, after the sensor spot is dried, the plasma treated acrylic lens has a sealing groove with a width of 2.7 mm, a height of 0.1 mm, and a length of 13.7 mm (total volume = 3.7 μl). The sample is then snapped onto the polystyrene substrate forming the sample port of the lens. is applied, which quickly fills the channel and reconstitutes the dried sensor.

[0095] In some embodiments, the cartridge is a microfluidic device and is made of polystyrene A substrate, an acrylic lens, a bilirubin-responsive probe, a non-responsive probe, and and a substrate having an anti-hemoglobin peptide immobilized thereon. In order to reduce the reflection intensity of the 660 nm excitation light, the polystyrene substrate is a dark gray substrate. In some embodiments, the substrate comprises a material having a dark color configured to: and / or anti-hemoglobin peptides attached to beads. In this method, the beads are applied to a defined area on a substrate as a slurry in a volume of less than 2 μL. In some embodiments, the substrate is The substrate polymer is treated with UV light having a wavelength in the range of about 145 nm to about 225 nm. In some embodiments, after curing (photo-treatment), the polystyrene polymer chains are photo-treated. The dry sensor forms a spot on the cartridge, such as a spot having a diameter of about 2 mm. In some embodiments, the lens is positioned on the base and guides the sample port through the sensor. The channel extends several mm beyond the base of the tube (Figs. 10A-10D, 11A-11D, and In some embodiments, the microfluidic device (cartridge) contains a desiccant The product is placed in a Steriflex W1F pouch containing:

[0096] 10A-10D show multiple views of one embodiment of the base plate of the cartridge. 1 shows a top view of the cartridge base 1000. The cartridge base 1000 has a substrate. The cartridge substrate 1000 includes an open area 1010 for holding the cartridge. and / or for inserting or removing the cartridge from the reader. The cartridge may further include a housing that may include ribs 1020 for The edge substrate 1000 can be made from any suitable material, including, for example, polystyrene. In some embodiments, the base 1000 can be a 660 nm excitation light source, such as a dark gray substrate. The material may include a dark color configured to reduce the reflected intensity of the light. In this state, the open areas 1010 of the substrate are treated with UV light having a wavelength in the range of about 145 nm to about 225 nm. In some embodiments, the light treatment renders the polystyrene polymer chains more susceptible to reaction with the substrate. In some embodiments, following the light treatment, the substrate is The substrate is contacted with the light-treated area of ​​the substrate, bonding the substrate to the substrate at specific areas within the substrate. , can include any of the substrates disclosed herein.

[0097] FIG. 10B shows an open area 1010 where a substrate can be placed and includes ribs 1020. FIG. 10C shows a side view of the cartridge base 1000. FIG. 10D depicts an enlarged cross-sectional view of the open area 1010, showing the base cart. Depicts ribs 1030 that are crushed when ridges 1000 are bonded to the lens.

[0098] 11A-11D show a lens configured to couple to a cartridge base 1000. 11A shows a multi-view of an embodiment of the sensor 1100. FIG. 11A shows a sample port 1105, a fluid flow path 1106, and a 11B shows a top view of the lens 1100 including the bilirubin sensor 1115. 11B shows a side view of the lens 1100. As shown in FIG. 11B, the lens 1100 is The port 1105 is coupled to the cartridge base 1000 to form a fluid flow path 1110; an insert 1120 configured to seal the cartridge to prevent fluid leakage; 11C shows a bottom view of the lens 1100 and fluid flow channel 1110. shows a cross-sectional view of a lens 1100 showing a sample port 1105 and a fluid flow path 1110.

[0099] The lens 1100 is adapted to be coupled to the open area 1010 of the cartridge substrate 1000. When mated, the lens is secured to the cartridge base ribs 1030. thereby sealing the cartridge, thereby removing the Furthermore, the lens 1100 is connected to the cartridge base 1000. By this, a fluid flow path 1110 is formed, and the fluid flows through the fluid flow path to the sample port 1105. and the fluid flows to the substrate.

[0100] In some embodiments, the fluid flow path is configured to include a specific Size and dimensions. In some embodiments, the surface-to-volume ratio of the fluid flow paths is large, It is sufficient to measure bilirubin in an undiluted sample. is used when the sample is undiluted whole blood, plasma, serum, urine, CSF, saliva, gastric juice, interstitial fluid, or lymphatic fluid. In some embodiments, the cartridge is a 0.1, 0. 2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, Less than 0.5 μL to more than 10 μL, such as 6, 7, 8, 9, 10, 15, or 20 μL It is configured to receive a range of sample volumes.

[0101] In some embodiments, the cartridge is configured to measure the sample at equilibrium. Conventional measurement devices and / or systems, such as those available commercially, are not in equilibrium. However, the cartridge of the present invention is capable of measuring lirubin at equilibrium. The device is configured to measure bilirubin in a sample.

[0102] In some embodiments, the cartridge enhances the accuracy of the bilirubin measurement through traceability. Traceability can be improved, for example, by comparing the probes described herein. The standard bilirubin sample obtained from Sigma was used to calibrate the Then, using the calibrated probe, bilirubin-human serum A set of albumin conjugates is calibrated. The calibrated conjugates are then used to The batch is calibrated (as described in more detail in the Examples herein).

[0103] Some embodiments provided herein include a cassette into which a sample-containing cartridge is inserted. The reader uses a 660 nm excitation light source to detect the sensor. By scanning across the sensor and measuring the fluorescence (background intensity) from before and after the sensor, However, the fluorescence intensity at 700 nm and 819 nm, including any fluorescence from the sample itself, is measured. The reader uses the measured fluorescence intensity to form an R value and calculates B using equation (2). Each cartridge is stored in a sealed pouch containing a desiccant. A barcode containing the calibration parameters is printed on the pouch and scanned by a reader. After applying the sample to the cartridge and inserting it into the reader, Bf appears in approximately 90 seconds. It can be shown.

[0104] Using the Sensor In some embodiments, the sample used to measure unbound bilirubin is human, animal or In some embodiments, the fluid is whole blood, plasma, serum, In another embodiment, the unbound bilirubin is present in urine, CSF, saliva, gastric juice, interstitial fluid, or lymph. The bottles are determined by microinjecting or otherwise transfecting the sensor into cells. This is done in the cytoplasm of cells by injecting the It is carried out in an external medium.

[0105] The range of unconjugated bilirubin may be determined from a healthy population, and from this normal range Deviations may indicate disease.

[0106] Non-binding bilirubin sensors with zero Rm are disclosed in U.S. Pat. No. 9,529,003 and Huber et al. Fluorescent sensor for quantification of unbound bilirubin concentration, Clin Chem 58: 869-876, 2012] It can be calibrated and used to measure [Bf] as described. The method of calibration and use of probes >0 is described herein by equations (1-3) (calibration) and Bf is first described in the calculation of equation (2).

[0107] The non-binding bilirubin sensor is used because the liver function is insufficient to eliminate excess bilirubin. Therefore, it is important to measure Bf in patients at risk for bilirubin-mediated toxicity, such as 80% of all newborns. [Bhutani et al., Pre-discharge screening for severe neonatal hyperbilirubinemia using photochromic angiography. Infants requiring radiotherapy are identified, J Pediatr 2013;162:47782].

[0108] The non-binding bilirubin sensor is suitable for patients with hemolytic diseases and those receiving intravenous infusion of oily emulsions. , patients receiving drugs that may displace bilirubin from albumin, and In cases of sepsis, which is common in premature infants, the binding affinity of bilirubin to albumin decreases, resulting in a decrease in FFA concentration. Patients with diseases in which Bf may increase due to increased blood pressure [Nogueira et al., in sepsis patients] Altered plasma free fatty acid concentrations in humans are associated with cardiac dysfunction and reduced heart rate variability. Shock 29 : 342-348, 2008, [Hegyi et al., Soybean lipid infusion increases unbound free fatty acids and and effects on unconjugated bilirubin, J Pediatr 2017; 184: 45-50].

[0109] Non-binding bilirubin sensors are used to reduce phototherapy, transfusion or bilirubin toxicity. It can be used for the measurement of Bf in patients undergoing other therapies as designed.

[0110] Because unconjugated bilirubin, not total bilirubin, is toxic, total bilirubin should be avoided during phototherapy. Unbound bilirubin is monitored rather than the bilirubin level, and unbound bilirubin is significantly reduced. Total bilirubin has been shown to decrease in response to phototherapy. However, in the presence of bilirubin-substituting molecules such as FFA and certain drugs prescribed to newborns, Under these conditions, total bilirubin and unbound bilirubin can be almost completely separated. Under these conditions, it would take virtually all the unconjugated bilirubin to reduce unconjugated bilirubin to a level that would be considered nontoxic. It is thought that complete destruction of bilirubin is necessary. This would require much lower total bilirubin levels than are currently achieved [He Gyi et al., Intralipid-treated preterm infants showed a significant increase in unbound free fatty acids from total bilirubin. Uncoupling of soluble free fatty acids may prevent phototherapy from working, Neonatology 2013; 1 04: 184-187 and Hegyi et al., Unconjugated free fatty acids and unconjugated bilirubin in preterm infants Effect of soybean lipid injection on the botulinum toxin, J Pediatr 2017; 184: 45-50]. Furthermore, this study The photoisomers of bilirubin or conjugated bilirubin and the "native" unconjugated IX-α(Z,Z) isomer Peroxidase assessment is not useful for monitoring unbound bilirubin during phototherapy because it does not distinguish between the active and the active forms. In contrast, the UBCheck sensor described in this application cannot be used to monitor The unconjugated bilirubin measured by the sensor is specific for the natural unconjugated IX-α(Z,Z) isomer. be.

[0111] The only method currently used to determine unconjugated bilirubin is the Western method of bilirubin. Based on horseradish peroxidase oxidation [Jacobsen J and Wennberg RP, in neonatal serum Determination of unconjugated bilirubin. Clin Chem 20: 783, 1974. Peroxidase assays are performed , available using FDA approved evaluation (Arrows Ltd, Osaka, Japan). The Arrows method [Ahlfors CE, plasma unbound unconjugated bilirubin measurement] complicates bilirubin measurement. Anal Biochem, 279: 130-135, 2000; Ahlfors et al., Unconjugated bilirubin in preterm neonates Influence of sample dilution, peroxidase co-feeding and chloride ions on the erythrocytes. Clin Biochem 40: 261- 267, 2007], jaundice is common in newborns. Importantly, multiple relatively large Plasma or serum with a suitable sample volume (20-25 μL) was analyzed using the Arrows UB analyzer UA-2. Furthermore, the Arrows evaluation, which involves diluting the sample 52 times, is required to measure Bf. Unbound bilirubin in premature infants was not determined using the Arrows method [Ahlfors et al. Effect of sample dilution, peroxidase concentration, and chloride ions on bottle measurements. Clin. ical Biochemistry 40 (2007) 261-267; Ahlfors et al., Unbound (free) bilirubin: a new Improved paradigm for assessing neonatal jaundice. Clinical Chemistry 55:7 1288-1299 (2009)] is required to correct for interfering substances.

[0112] The embodiments provided herein provide a method for measuring unbound bilirubin in a single step. In some embodiments, the method overcomes the drawbacks of the Peroxidase Arrows method. In some embodiments, the method overcomes equilibrium non-binding in undiluted blood samples. Fluorescently labeled mutant fatty acid binding protein allows direct monitoring of bilirubin levels The probe detects the Z,Z isomer of unconjugated bilirubin. It is specific to the Z,Z isomer of bilirubin and can bind with high affinity to it. In addition, the unbound bilirubin sensor can be highly specific for unbound bilirubin and free bilirubin. Fatty acids (FFA), bilirubin photoisomers, conjugated bilirubin, other metabolites, and blood Unresponsive to or not significantly binding to most drugs present in the The Vincensor assesses the potential risk of bilirubin neurotoxicity and thereby the risk of such toxicity. In order to accurately direct treatment to prevent non-conjugation in jaundice patients, including newborns, It can be used to determine combined bilirubin levels.

[0113] In some embodiments, the method comprises determining whether bilirubin is present in the brain based on the results of the risk of bilirubin neurotoxicity. Subjects selected or identified as having or suffering from Rubin neurotoxicity In some embodiments, the method further comprises administering a treatment or therapy to the patient. Treatment may include phototherapy, exchange transfusion, intravenous immunoglobulin therapy (IVIg), or bilirubin In some embodiments, the phototherapy treatment or therapy is a blue-green inhibitor. This includes exposure to lamps that emit light across the color spectrum, and light exposure increases bilirubin excretion In some embodiments, exchange transfusion involves repeatedly withdrawing blood and transferring it from a donor to a recipient. In some embodiments, this includes replacing the affected blood with unaffected blood, such as blood from a patient. The study was conducted to determine whether IVIg treatment or treatment resulted in the development of antibodies in the blood of subjects suffering from bilirubin neurotoxicity. Other treatments or therapies include intravenous transfusion of blood proteins that can lower blood levels. Therapies include, for example, treatment or inhibition or amelioration of bile duct obstruction, infectious causes or genetic disorders (e.g. Bilirubin, including the treatment or inhibition or amelioration of inflammatory bowel diseases (e.g., Crigler-Najjar and Gilbert syndromes) This may involve treating or inhibiting the underlying cause of bin neurotoxicity. [Example]

[0114] Embodiments are further defined in the following examples, which are provided for illustrative purposes only. From the above discussion and these examples, it should be understood that Those skilled in the art will be able to ascertain the features of the embodiments described herein and will understand the spirit and scope of the invention. Various changes and modifications may be made to the embodiments without departing from the scope thereof, making them suitable for various applications. and conditions. In addition, various modifications of the embodiments will become apparent to those skilled in the art from the foregoing description. Such modifications also fall within the scope of the appended claims. The disclosures of the references are incorporated herein by reference in their entireties and are incorporated herein by reference. For disclosure purposes.

[0115] Example 1 Produced by mutation of wild-type intestinal fatty acid binding protein (SEQ ID NO: 1) Sequences of bilirubin-sensitive (responsive) and non-responsive probes Wild-type intestinal fatty acid binding protein (WT rIFABP) from rat is identified as SEQ ID N Table 1 shows the single residues at positions 24 to 98 of SEQ ID NO:1. Bf-sensitive probe with LICOR700DX-maleimide labeled with recombinant cysteine Furthermore, each probe listed in Table 1 has an N-terminal MGI. The probes are 7R, 16R, 20R, 29R, 37R, 46R of SEQ ID NO: 1, 50R, 88R, 92R, 94R, 100R, 125R, 129R and 130R (KR 14) has 14 accessible lysine to 14 arginine substitutions. The sequence of is shown in SEQ ID NO: 2. Responsive and non-responsive mutants have a C-terminal double HI S-tag linker, Arg Gly Ala Ala Ser His His His His His His Ser His Arg Ala Thr Pro Asn Thr Ser P ro His His His His His His (C2XH11; SEQ ID Therefore, each probe listed in Table 1 has the additional sequence shown in the table. In addition to substitutions and additions, it contains the N-terminal MGI-KR14-C2XH11.

[0116] [Table 1]

[0117] [Table 2]

[0118] Example 2 Influence of non-fluorophore position on Bf binding properties and emission spectra of non-responder proteins Non-mutational effects on Tables 1 and 2: LICOR70 labeled with LICOR800CW-maleimide probe All responding probes labeled with 0DX-maleimide and non-responding probes were and have a qualitatively similar dependence on Bf. The probes are used to measure protein expression, binding, and They differ mostly in parameters (Kd, Ro and Rm) and stability. The effects of different mutant proteins and fluorophore labeling positions on Rm were investigated. A low Kd and a low responsive probe indicate the degree to which the probe is quenched by bilirubin binding. and / or have low Qs (Qs=Rm / Ro) (Table 3). Non-responders are not characterized by bilirubin binding parameters, but The emission spectrum of LICOR700DX-maleimide can be changed by changing the intensity. For example, in the absence of 700DX-maleimide, many Early non-responsive mutants reveal a time-dependent increase in emission at 700 nm relative to 819 nm In some of these mutants, the 700-819 nm ratio decreased during storage at 4°C. The instability increased to about 1%-20%. By appropriate mutations, this instability was eliminated. For example, Mut:SEQ ID NO:1 B C73 in Table 2 has the same 700 / The ratio was 819 (1%).

[0119] [Table 3]

[0120] Example 3 Changes in fluorescence of bilirubin-responsive (700 nm) and non-responsive (800 nm) probes and and their ratio (700 / 800) Figure 1 shows the bilirubin-responsive LICOR700DX maleimide Mut: SEQ ID NO: 2-76C probe (Table 1) and bilirubin-unresponsive LICOR800CW maleimide Mut:SEQ ID NO:1 Bf sensor consisting of B-73C probe (Table 2) The intensities and intensity ratios of the α- and β-glucan derivatives are shown, both of which are free in aqueous buffer and are predominant at the concentrations used. The total bilirubin (BT), which is unbound bilirubin (Bf), increases. Measurements of the 710 and 805 nm intensities were performed using a Horiba Fluorolog2 at 1.5 nm and 1.5 nm, respectively. The results were obtained using probe concentrations of 10 nM and 10 nM. Monotonic decrease in LICOR700DX maleimide intensity (measured at 710 nm), and non-responsive iL The lack of intensity change (cv) of LICOR800CW maleimide attached to BP (measured at 805 nm) = 2.5%). The inserts show a behavior of the 710 / 805 ratio.

[0121] Example 4 Calibration of free NIR ratio sensors by cuvette fluorescence method LICOR700DX maleimides from Tables 1 and 2, respectively, released in aqueous buffer. Doped-labeled bilirubin-sensitive iLBP (Mut: SEQ ID NO: 2-76C) and LI COR800CW maleimide-unresponsive iLBP (Mut: SEQ ID NO: 1B-73 C) was used to measure the Bf sensor consisting of Sigma bilirubin (Cat. The BT concentration was titrated at increasing concentrations from a stock solution of BT (B4126) (Figure 2). At each bilirubin concentration, the fluorescence excited at 660 nm and Measurements of emission at 700 nm and 805 nm were used to determine the 700-805 nm peaks from the background. The ratio (R) of the total bilirubin B in the cuvette at each step of the titration was determined. T is the total free bilirubin excluding the portion that is soluble and binds to the sensor. A least-squares fit to the curve (R vs. BT) (Figure 2) was performed using the equation that takes into account probe-bound bilirubin. (1) In this procedure, the calibration parameters (Kd, Rm, and Ro) are Calibrate the free probe by determining the Kd of 0.4 ± 0.4 nM, Rm of 0.42 with Ro of 3.02, and 0.139 ± 0.0 It was Qs, Rm / Ro of 07.

[0122] Example 5 Calibration of bilirubin-albumin (HSA) complex To calibrate the Bf test cartridge, a highly buffered Bf concentration was added. A series of aqueous samples are prepared. A highly buffered Bf sample is prepared whose Bf concentration is Bilirubin is released by binding to the walls of the cleft and also by binding to a dried bilirubin probe in the cleft. Bilirubin bound to HSA (BR:HSA) and 400- Complexes with HSA at a concentration of 1000 μM were calibrated for negligible bilirubin wall binding. The Bf concentration in the test cartridge remains unchanged from the level at which it was buffered or The BR:HSA complex typically exhibits a 0.05-0.1 step size. The complex is prepared in a cuvette with a molar ratio of bilirubin to HSA of 0.1 to 1.0. The Bf concentration produced by each conjugate was calibrated as in Example 4 for each undiluted conjugate. Calibration was performed by adding approximately 1-20 nM of the free Bf sensor. Typically, Bf values ​​increase exponentially as BR:HSA increases from 0 to 0.9. The Bf increases functionally, starting from 0.1 and increasing from 2 to 300 nM (Figure 3 ).

[0123] Example 6 Calibration of test cartridges with BR:HSA complex A quantity of products manufactured in a given period, called a "lot," for example, 500 to 2000 or more. A significant number of test cartridges are used, each containing a bilirubin-albumin complex. Comparisons were performed by randomly selecting a sufficient number of cartridges so that the A single calibration of a lot requires at least three measurements of each complex, which Depending on the BR:HSA step size, the range is 33 to 60 cartridges. Cartridges are used for a single measurement and then discarded. The measured R value from the formula is used to obtain the Ro, Kd, ​​and Rm for the cartridge lot. The data were fitted by least squares using BR:HSA complex. An example of cartridge calibration is where the apparent Kd and Qs are similar to the free probe parameters. It is clear that the increase in Kd can be as high as or even higher than that (Figure 3). Immobilization of probes with adjacent surfaces during drying of probe-beads on the cartridge and interactions.

[0124] Example 7 Single-Step Disposable Cartridge The single-step disposable cartridge is an essential component of the present invention. When adding a blood sample and inserting the cartridge into the reader, all the information required to measure Bf is The weight of a newborn baby is only about 400g, which is too small to measure Bf. It is essential to use a blood volume. The minimum sample volume for the disposable cartridge is 3.8 μl. Furthermore, since whole blood, as well as plasma, serum and other fluids, are measured, the excitation light path and The optical path lengths of the inlet and outlet optical paths must be as short as possible. These considerations are reflected in Figure 10A. This led to the development of the cartridges shown in Figures 10A-10D and 11A-11D. The sample is added to the port and the cartridge contains a bilirubin sensor spot in the center of the groove. Fill the groove formed by the acrylic lens snapped onto the base. The grooves are 0.1 mm high, 2.7 mm wide, and accommodate a sample volume of 3.8 μL. It has a length of mm.

[0125] Maintaining these dimensions requires high tolerances in molding. The essential and novel component is a clear acrylic lens simply snapped onto a polystyrene base. By doing so, a sealing groove was formed on the dried Bf bilirubin sensor-polystyrene beads. The ability to form a cellular membrane is essential for the formation of membrane-like structures (Figures 10A-10D, 11A-11D, and 12). When the lenses are snapped into place, they create an oval groove in the base, which fits over the surface of the base. The sealed channel is formed by pressing the material against the crushing rib underneath the surface. This is achieved by achieving rapid capillary flow of the sample over the bilirubin sensor spot. Therefore, acrylic lenses can be used to reduce the amount of light emitted from the lens without compromising its optical clarity in the NIR. It is treated with O2 plasma at a level that increases its hydrophilicity. It increases its hydrophilicity, enhances rapid capillary flow, and adjusts the sensor spot size. The UV irradiation also acts to remove the polystyrene of the bilirubin sensor. This is important to allow the beads to firmly attach to the polystyrene substrate. UV irradiation at 54 nm breaks the polymer bonds, thereby freeing the polymer chains on the opposing surfaces. It has been shown to allow intercalation of cations and increase the binding affinity between surfaces. [Maeda et al., Adhesion and friction mechanisms of polymers on polymer surfaces, Science (2002) 297 , 379382]. Irradiation from a 254 nm source increases the binding affinity between polystyrene surfaces. However, the degree of adhesion is very sensitive to the life of the 254 nm bulb, and the irradiation time The present invention also provides a typical 254 nm bulb that can be used for 18 minutes or longer. The discovery that 185 nm radiation produces low levels of 5 nm radiation, and that 185 nm radiation produces increased potency. This suggests that switching to a 185 nm light source is the main cause of polymer adhesion. Depending on the application, exposure times can be reduced to seconds or minutes. By spotting the sensor on the ridge, a more uniform spot shape can be obtained. Thus, in some embodiments, wavelengths of 145, 150, 155, 160, 165, 1 70, 175, 180, 185, 190, 195, 200, 205, 210, 215, 2 20, or 225 nm, or a range of about 145 nm to about 225 nm, or any of the foregoing values It is a wavelength within the range defined by any two of them.

[0126] The Bf sensor attached to the cartridge surface is described in U.S. Pat. No. 9,529,003. In contrast to the fully quenched sensor, it is incompletely quenched by bilirubin For example, U.S. Patent No. 9,529,003 describes a method for immobilizing a nucleotide sequence immobilized in the wells of a "disposable cartridge." , with a HIS tag and a polystyrene tag (PS) attached to Ni-agarose beads describes a bilirubin probe that can be used in the detection of bilirubin, but does not provide details of such a cartridge. As shown in Figure 8 of Patent No. 9,529,003, the Qs (Rm / Ro) of the probe is 0.02±0 0.03 (Rm=0), indicating a fully quenched Bf sensor. The limit of extinction (Rm>0) uses a different assay to determine the exact Bf level. The analysis described herein is mathematically detailed in Equations 1 and 2, which show that for RM=0 This is reduced to equations 2 and 3 of US 9,529,003.

[0127] U.S. Patent No. 9,529,003 uses a magnet to measure B on iron-containing polystyrene beads. f In a microfluidic device where sensors are concentrated at the bottom of a multiwell plate, This describes the measurement of Bf in blood. This configuration uses a sample cartridge containing a desiccant. The cartridge must be sealed in a pouch so that the sensor is tightly bonded to the cartridge surface. Instead, in this disclosure, polystyrene beads are The slurry containing bilirubin-sensitive and -unresponsive iLBP mutants attached to the slurries was were prepared, each with a different NIR fluorophore labeled with a cysteine ​​residue, All in aqueous buffer. A small drop (<2 μl) of this slurry was placed on a UV-treated cartridge. The mixture is dispensed into a desiccant containing pouch, dried, and placed in a desiccant containing pouch.

[0128] Example 8 Determination of equilibrium Bf concentration Bf in a blood sample is in equilibrium due to the binding and dissociation of bilirubin and albumin. The typical albumin concentration is 400-600 μM, which is about 100% for adult human albumin. The equilibrium dissociation constant (Kd) for all bilirubin is approximately 20 nM for the high affinity site. At equilibrium, a bilirubin-albumin molar ratio of 0.5 produces approximately 20 nM Bf. The estimated capacity of the Bf sensor on the ridge is 2x10 -13 Therefore, in a 5 μl sample, The sensor concentration is 40 nM. This means that at best, the sensor can detect bilirubin below 40 nM, is approximately 1.6 x 10 of 250 μM total bilirubin bound to 500 μM albumin. -4 Combine with This means that Bf is approximately Kd*BT / albumin, so a small change in BT has a negligible effect on Bf. The actual albumin buffer Bf equilibrium concentration Since the effect of the sensor on the concentration is negligible, UBCheck approximates the equilibrium Bf concentration. This method allows the bilirubin-albumin complex to be detected by the sensor without diluting the sample. This produces highly buffered Bf levels that remain undisturbed by the amount of Bf bound. Another method for measuring Bf in blood samples is the equilibrium measurement. Most importantly, it is used in the 45-year-old, FDA-certified ArrowsUB analyzer. The peroxidase method currently practiced does not produce Bf at equilibrium [Jacobsen et al., New Determination of unbound bilirubin in plasma of newborns, Clin Chem (1974) 20, 183]. This is due, in part, to The large sample dilution factor (42-52 times) in the FDA-approved Arrows method and the substantial reduction of Bf The method is based on the peroxidase method, which oxidizes the fraction. By measuring the concentration of Bf and extrapolating to zero peroxidase concentration, A better estimate can be obtained [Ahlfors et al., Unbound (Free) Bilirubin: Neonatal Jaundice]. Improving the paradigm for assessing chemoattractants, Clin Chem (2009) 55:7 1288-1299]. However, this method is not FDA approved and will ultimately result in a better equilibrium approximation. However, without measuring the equilibrium, the results obtained by the present invention or the peroxidase method were More recent methods have been proposed for measuring Bf, yielding Bf concentrations several orders of magnitude higher than [Bell et al., Paper-based potentiometric detection of free bilirubin in serum, Biosensors and Bioelectronics, 126(2019)115-121]. This method uses a filter and electromotive force. By separating bilirubin from albumin using Destroy.

[0129] Example 9 Effect of dilution on equilibrium - bilirubin-albumin substitution increases HSA Kd case UBCheck provides the most accurate measurement of equilibrium Bf and thus steady-state Bf levels in circulation. The direct explanation for the buffering capacity of the bilirubin:albumin complex is The response of the sensor in the cartridge with and without albumin is shown in Figure 1. Addition of free bilirubin at a concentration of 0.00 nM resulted in a ≤1% Bf concentration as detected by the Bf sensor. When 1000 nM of free bilirubin is added, 50 nM of Bf is detected. This loss of bilirubin is particularly pronounced in the cartridge due to the large surface-to-volume ratio of the sample groove (20). Define the sample groove within the polystyrene and acrylic surface.

[0130] Even without surface binding, the equilibrium Bf concentration is due to the kinetics of the bilirubin-HSA reaction. It depends on the sample dilution. Furthermore, the effect of dilution on the binding affinity of HSA for bilirubin This is amplified in the presence of a substance that effectively reduces bilirubin by displacing it from albumin. , when oleic acid was added to HSA at a molar ratio of 6 oleic acid to 1 HSA, The results are shown in the measurement of Bf as a function of bilirubin-HSA complex at a molar ratio of 0.5. Starting with a 550 μM HSA concentration, the Bf concentration was increased by a 42-fold dilution (the original Arr The dilution used by the owsUB analyzer reduces the concentration by more than an order of magnitude. Oleic acid is a potent F It is a FA substitute and the main component of Intralipid, a drug commonly prescribed for premature infants in the NICU. .

[0131] Example 10 Hemoglobin / hemolysis has no effect on UBCheckBf measurement After applying Bf-responsive and non-responsive probes to Ni-polystyrene beads, The 4 peptide was added to the beads and attached to the Ni-NTA of the beads through the double HIS tag. The combined probe and peptide beads are then spotted onto the cartridge. The effect of whole blood hemolysate was examined by adding bilirubin to neonatal blood samples at increasing hemoglobin concentrations. The Bf concentration was measured before and after hemoglobin titration and the zero The Bf concentration relative to hemoglobin is at least as high as 4 g / l. We found that the effect of α-glucan on the oxidative stress was not affected by the α-glucan content (Figure 5).

[0132] [Table 4]

[0133] Example 11 Effects of medications prescribed for newborns The embodiments described herein relate to measuring the concentration of Bf in newborns. Premature infants in NICUs are at high risk for bilirubin neurotoxicity because they receive frequent medications. are of particular concern. [Hsieh et al., Drug Use in Neonatal Intensive Care Units, Am J Perinatol (2014) 31, 811-822]. Table 5 shows the specific medications most frequently prescribed in the NICU. As determined in the measurement of Bf using the one-step cartridge of Example 7 , which has been shown to be a potent displacer of bilirubin from albumin and interference. The most prescribed drug in the NICU was ampicillin, which was ranked #1. The elements in the "Substitution or Interference" column indicate whether the drug has no effect or whether it is a brand. The displacer was positive, with a 3-fold increase in drug concentration compared to no drug. The negative values ​​for spironolactone indicate interference with the Bf sensor. As such, some drugs are potently substituted, and spironolactone may interfere with Bf assessment. It is the only NICU drug found to have a beneficial effect on the release of bilirubin from albumin. Function of prescribed (low, medium, high, and 3x high) drug concentrations for drugs in Table 5 that are potent substitutes The effect on Bf as a

[0134] [Table 5]

[0135] The increase in Bf caused by these drugs may greatly exceed the upper limit of normal in NICU infants. This may have serious consequences for their health. The increase in Bf caused by these drugs may greatly exceed the upper limit of normal in NICU infants, and Furthermore, FFAs such as those described in Example 7 may have serious health consequences. The effect of dilution, as well as the effect of oleic acid, depends on the degree of displacement caused by the drug. Table 6 shows the effect of bilirubin, followed by cefazolidin at 1.12 and 3.76 mM. We demonstrate this effect by comparing Bf levels in adult serum spiked with chloramphenicol. The determination was performed using the Arrows UB2 analyzer (52-fold dilution) and UBCheck (undiluted). As shown in Table 6, Bf in the presence of 1.12 and 3.76 mM cefazolin was ,In UBCheck, it is about 3 and 5 times larger than Arrows.

[0136] [Table 6]

[0137] Example 12 Reduced interference from conjugated bilirubin Determine the effect of ditaurobilirubin on UBCheck's ability to accurately measure Bf This will determine potential interference between conjugated bilirubin and the bilirubin probe. The results of UBCheck were compared with measurements using the Arrows UB analyzer UA-2 method. Neonatal serum was spiked with unconjugated bilirubin to obtain a Bf of approximately 10 nM, and then spiked This is done by titrating the sample with ditaurobilirubin to 20 mg / dL (Figure 7). During the process, Bf was measured by UBCheck and Arrows, and direct (bound) bilirubin concentration was measured. The level is determined using the Sigma direct bilirubin kit. Both UBCheck and Arrows yield 10 nM Bf, while direct bilirubin measurement yields approximately 0.5 mg. / dL yields non-zero values ​​(Figure 7). As the concentration of ditaurobilirubin increases, UBCheck did not change, but Arrows started at about 0.5 mg / dl ditaurobilirubin and increased. Then, ditaurobilirubin of 4 mg / dl or higher was added, at which the Arrows evaluation saturated at Bf ≥ 50 nM. In contrast, UBCheck increased only at ditaurobilirubin levels of 4 mg / dl. Initially, the Bf increased to approximately 12 nM, a 20% increase from the initial 10 nM level, and Only at 20 mg / dl ditaurobilirubin does Bf increase to 33 nM. Check outperformed Arrows for Bf even when the Arrows sample was diluted 52-fold. Although the UBCheck sample has excellent specificity, it is not diluted.

[0138] Example 13 Lack of interference from bilirubin photoisomers Neonatal hyperbilirubinemia is caused by blue-green photoisomerization of bilirubin bound to albumin. It is most frequently treated with color phototherapy. [Newman et al., Neonatal Hyperbilirubinemia and Long-Term Hyperbilirubinemia] Outcomes: Another observation from the collaborative perinatal project, Pediatrics (1993) 92, 651-657, Enn ery JF, Blue light, green light, white light, and other lights: Treatment of neonatal jaundice. Clinical practice in perinatal medicine. 1990)17, 467-481]. Bilirubin photoisomers (4Z,15E), (4E,15Z) and Lumirubin is much more soluble and therefore more easily absorbed than the natural bilirubin IXa (4Z, 15Z) molecule. Phototherapy readily reduces the Z,Z isomer, thereby reducing the Effectively treats neonatal hyperbilirubinemia. Photoisomers are also produced by exposure to ambient light. Photoisomers: A potential role in clinical peroxidase assays of unconjugated bilirubin [McDonagh et al. Confounding factor? Pediatrics (2009) 123, 67-76]. As shown in Figures 9A and 9B, The valence detects only the Z,Z isomer of bilirubin, whereas the Arrows detects Z,Z and all photoisomers. However, only Z,Z is toxic, not the photoisomers [Jasparova et al., Biological Photoisomers of Lirubin (2016) PLoS ONE 11(2):e0148126.doi: 10.1371 / journal.pone.01 48126]. Although no phototherapy was administered, HPLC analysis revealed significant levels of photoisomers (probably Serum samples from healthy newborns showing oxidative stress (due to ambient light exposure of serum) were also analyzed, as shown in Figure 9A. , Arrows show more than double the Bf levels compared to UBCheck Bf assessment. Figure 9 As shown in B, adult serum samples were spiked with bilirubin and then exposed to a phototherapy lamp (Natus The top panel shows multiple HPLC runs over the 5-hour exposure. The relative intensities of Z, Z, and the three photoisomers determined from the can are shown. decreases monotonically to a low level, while the photoisomers peak at about 1.5-2 hours and then The bottom panel shows two samples spiked with bilirubin. The results show that Bf levels were 8 days before phototherapy by both Arrows and Bf assessment. In contrast to the arrows, the Bf estimates showed a monotonic decrease of Z,Z towards zero. Meanwhile, ArrowsBf rises rapidly until it reaches its saturation level of 50 Thus, Arrows is highly sensitive to photoisomers.

[0139] Bleaching serum with a phototherapy lamp as shown in Figure 9B showed that after 5 hours, the serum was Such experiments reveal that the Bf concentration is essentially zero. limits of quantitation, and furthermore, they allow for the detection of U in serum or plasma lacking bilirubin. If BCheck contains other hydrophobic metabolites such as fatty acids, other lipids, peptides, nucleic acids, etc. Importantly, this demonstrates that the blood-borne markers are not affected by other blood-borne molecules. Phototherapy treatments are typically used for much longer periods (typically 24-72 hours). Given this, it is possible that the light from phototherapy lamps may be destructive to other blood components. The zero response tendency of UBCheck means that the evaluation is based on FFA, peptide, Insensitive to all natural blood metabolites, including nucleic acids, or any other natural blood components. UBCheck is not only insensitive to photoisomers, but also to the soluble form of the compound, demonstrating that it is not sensitive to photoisomers.

[0140] Example 14 Lack of interference from intralipids and triglycerides 8A-8B show that the lipids can be converted into triglycerides, which can reduce Bf (Fig. 8 ), which increases Bf by displacing bilirubin from albumin during lipolysis. In Figure 8A, 24 nM of FFA was generated, which can be used to treat inflammatory bowel diseases (Figure 8B). Neonatal serum samples spiked with bilirubin to yield a Bf of 1000 were spiked with triglycerides up to 10 mM. Titration with Intralipid resulted in a monotonic decrease of Bf to 18 nM. The results of these studies seem to correspond to intralipid infusion without heparin. The decrease in Bf was due to the UBCheck method. As a result, triglycerides create a sink for bilirubin. , and therefore is not an interfering substance. Figure 8B shows that in the presence of heparin, 1 g / kg / day (IL1) Intralipid infusions starting at 2g / kg / day (IL2) and up to 3g / kg / day (IL3) The results are shown for approximately 100 premature infants who underwent intralipid therapy. The heparin-activated release of unbound FFA (FFAu) displaces bilirubin from albumin. These results suggest that the Bf measurement is related to the triglyceride or This is consistent with the absence of FFA interference. In other words, turbidity (light scattering) is due to at least the following reasons: Reason: 1) Fluorescence excitation (660 nm) and emission (700 and 819 nm) are in the NIR (wavelength 2) the optical path length of the cartridge is 0.1 mm. and 3) if the 700 / 819 ratio is affected by scattering, the 700 intensity will be 8 19 intensity, thereby decreasing the R value, and thus the Since the ratio increases rather than decreases, In contrast, the peroxidation performed by Arrows The ELISA method (measured at 460 nm) showed that Arrows increased with increasing intralipid levels. The increase in total bilirubin is highly affected by scattering.

[0141] Example 15 Lack of interference from FFAu Lipid injections, such as interlipid in the presence of heparin, produce exceptionally high concentrations of unbound F. They can produce FAs (FFAu), many of which displace bilirubin from albumin. In neonates receiving increasing concentrations of Interlalipid, the FFAu levels The level can increase by more than 100 nM [Hegyi T. et al., Soybean lipid infusion in premature infants Effects on conjugated free fatty acids and unconjugated bilirubin, (2017) J Pediatr 184, 45-5 However, the results in Figure 4 show that when the molar ratio of oleic acid to albumin is 6:1, , unbound oleic acid concentrations greater than 500 nM [Richieri et al., Long-chain fatty acids and albumin Interaction of amines: Measurement of free fatty acid levels using the fluorescent probe ADIFAB, Biochemistry 32 7574-7580 (1993)], but by increasing Bf, bilirubin can be converted to albumin. It is clear that the FFA does not interfere with the Bf sensor, since only the increase in the It is set to.

[0142] Example 16 Analytical specifications and determination of Bf in bilirubin-spiked and non-spiked human serum / plasma Following CLSI guidelines, the analytical specifications for UBCheck evaluation were The results were determined using a kit and two or more readers. Limit of blank (LOB = 0.7 nM), limit of detection (LOD = 0.9 nM) determined by and limit of quantitation (LOQ). A precision test was performed at three centers using serum samples from newborns and adults spiked with riboflavin and bilirubin. The results, as an average of three reader and cartridge lots, are as follows: UB levels (nM) and CV (%): 4.4, 9%; 8.0, 7%; 11.4, 8%; 19 .1, 7%; 38.2, 8%).

[0143] Pooled human plasma with an albumin concentration of 620 μM (Golden West Biologicals) Bilirubin was spiked into the blood to produce a blood sample with a bilirubin / albumin molar ratio of approximately 0 to 0.9. Plasma samples were generated. [Bf] measurements were also performed using a peroxidase assay on an Arrows Bf analyzer. These results indicate that the bilirubin probe response is complete. The bilirubin probe was found to be present in human blood samples. Furthermore, the peroxidase method and Agreement with the bilirubin-albumin equilibrium predictions also indicates that the probe accurately measures unbound bilirubin. Demonstrate that the concentration is produced.

[0144] As used herein, section headings are for organizational purposes only. and should not be construed as limiting the described subject matter in any way. including, but not limited to, licenses, patent applications, papers, books, articles, and internet web pages. All literature and similar materials cited in this application, without limitation, are expressly incorporated herein by reference. and the disclosures specifically referenced therein are expressly incorporated by reference in their entirety for any purpose. The definitions of terms in the incorporated references are provided in this disclosure. In the event that definitions appear to differ from those provided herein, the definitions provided in this disclosure shall control. Minor deviations and slight variations in temperatures, concentrations, times, etc. discussed in this disclosure are to be considered. The presence of an implied "about" so that deviations are within the scope of the present disclosure herein is understood.

[0145] In this application, the use of the singular includes the plural unless otherwise specified. "comprises," "comprising," "contain" "s")," "contains," "containing," and "include" are used to express limitations. It is not intended to.

[0146] As used in this specification and claims, the singular forms "a," "an," and "the" " includes plural references unless the content clearly dictates otherwise.

[0147] While the present disclosure has been described in the context of specific embodiments and examples, those skilled in the art will recognize that the present disclosure may be practiced in various ways. Other alternative embodiments and / or uses thereof beyond the specifically disclosed embodiments, and It will be understood that the present invention covers obvious modifications and equivalents thereof. Although variations have been shown and described in detail, other modifications that fall within the scope of this disclosure may be made based on this disclosure. Various specific features and aspects of the embodiments will be readily apparent to those skilled in the art. Various combinations or subcombinations may be made and still fall within the scope of this disclosure. It is also contemplated that various features and aspects of the disclosed embodiments may be incorporated into various models. may be combined or substituted for one another to form modes or embodiments. It should be understood that the scope of the disclosure described herein is therefore limited to the above. It is not intended that the present invention be limited to the specific disclosed embodiments. .

[0148] However, while this detailed description sets forth various embodiments, it is understood that various modifications and variations within the spirit and scope of the present invention are within the scope of the present invention. Various changes and modifications will be apparent to those skilled in the art and therefore the following description is given by way of example only. Please understand that.

[0149] The terms used in the description presented herein should not be construed in a restrictive or limiting manner. Rather, the term simply refers to systems, methods, and related components. It should only be used in conjunction with the detailed description of the preferred embodiment. It may contain several novel features, only one of which alone contributes to its desirable attributes. are not responsible for or required to implement the embodiments described herein. It is not thought that there is.

Claims

1. 1. A sensor for measuring free bilirubin in a sample, the sensor comprising: a bilirubin-responsive probe labeled with a first fluorophore, and a non-responsive probe labeled with a second fluorophore; Here, the first and second fluorophores are excited at the same wavelength, and the first and second fluorophores are The sensor is made up of fluorescent dyes whose fluorophores fluoresce at different wavelengths.

2. The bilirubin-responsive probe comprises a first intracellular lipid-binding protein (iLBP). the first iLBP having a peptide sequence comprising SEQ ID NO: 1; Ar replacing 14 accessible lysines (KR14 as set forth in SEQ ID NO: 2) g; A C-terminal double His tag linker (C2XH) having the sequence set forth in SEQ ID NO: 3 １１）； N-terminally added MGI and Up to 62 amino acid substitutions and additions, including a single cysteine; The sensor of claim 1 , comprising:

3. The bilirubin-responsive probe comprises the sequence of any one of the probes listed in Table 1. The sensor according to any one of claims 1 to 2.

4. The non-responsive probe comprises a second iLBP, the second iLBP having SEQ ID N O:1, substitutions at positions 72, 73, 74, 126, and 131; C at any one of positions 27, 31, 33, 54, 73, 74, 76, or 98 Substitution to ys; no more than three additional amino acid substitutions; and A C-terminal double His tag linker (C2XH) having the sequence set forth in SEQ ID NO: 3 １１）； The sensor according to any one of claims 1 to 3, comprising:

5. 10. The method of claim 1, wherein the non-responsive probe comprises the sequence of any one of the probes listed in Table 2.

5. The sensor according to any one of claims 1 to 4.

6. The first fluorophore and the second fluorophore are different fluorophores. The sensor according to any one of claims 1 to 5.

7. The bilirubin-responsive probe comprises a single cis-fluorophore attached to the first fluorophore. The sensor of any one of claims 1 to 6, comprising a tain.

8. The non-responsive probe contains a single cysteine ​​to which the second fluorophore is attached. The sensor according to any one of claims 1 to 7, comprising:

9. LICOR 700DX Malay, wherein the first fluorophore is attached to a cysteine ​​substitution.

8. The compound according to claim 1, wherein the compound is LICOR800CW maleimide or LICOR800CW maleimide. Sensor.

10. The bilirubin-responsive probe binds to the unconjugated IX-α(Z,Z) isomer of bilirubin. The sensor of any one of claims 1 to 9, configured to couple.

11. The bilirubin-responsive probe binds minimally to conjugated bilirubin (less than 4 mg / dl). The sensor according to any one of claims 1 to 10, configured to:

12. The bilirubin-responsive probe is a probe that reacts with the Z,E or E,Z photoisomer of bilirubin and luminescence. Rubin, fatty acids, any other naturally occurring blood components, and / or neonatal drugs The sensor of any one of claims 1 to 11, configured to be non-binding.

13. The sensor of claim 12 , wherein the neonatal drug is not spironolactone.

14. The non-responsive probe is a probe that binds to the unconjugated IX-α(Z,Z) isomer of bilirubin or the conjugated IX-α(Z,Z) isomer of bilirubin. The sensor according to any one of claims 1 to 13, which does not bind to lirubin.

15. the non-responsive probe comprises the Z,E or E,Z photoisomer of bilirubin, lumirubin, Does not bind to fatty acids, any other naturally occurring blood components, and / or neonatal drugs The sensor according to any one of claims 1 to 14, configured as follows:

16. When the first fluorophore is LICOR700DX maleimide, The fluorophore in 2 is LICOR800CW maleimide attached to a cysteine ​​substitution. and when the first fluorophore is LICOR800CW maleimide, 16. Any one of claims 1 to 15, wherein the second fluorophore is LICOR700DX maleimide.

1. The sensor according to claim 1.

17. wherein the first fluorophore or the second fluorophore is attached to a cysteine ​​substitution; The cysteine ​​substitutions are 22, 24, 25, 26, 27, 29, 3 of SEQ ID NO: 1 Any one of claims 1 to 16, wherein the amino acid is at position 0, 33, 54, 74, 76, 97, or 98 The sensor according to claim 1.

18. The emission intensity of the first fluorophore or the second fluorophore is determined based on the bilirubin and hemoglobin, 18. A sensor according to any one of claims 17 to 17.

19. The bilirubin-responsive probe or the non-responsive probe contains at least one phosphoryl group. The sensor of any preceding claim, further comprising a car.

20. A composition comprising the sensor according to any one of claims 1 to 19.

21. A composition comprising a free bilirubin (Bf) sensor, the Bf sensor detecting bilirubin A first intracellular lipid-binding protein (iLBP) is bound to the IL-111A-binding protein and labeled with a first fluorophore. ) and a second iLBP that does not bind to bilirubin and is labeled with a second fluorophore. Including, wherein the second fluorophore is not bound to the first iLBP, and the first fluorophore and The first and second fluorophores are excited at the same wavelength, and the first and second fluorophores are excited at the same wavelength. The emission wavelengths of the two fluorophores are different, and the second fluorophore changes its emission in the presence of bilirubin. Does not change composition.

22. The first fluorophore is LICOR700DX maleimide, and the second fluorophore is the fluorophore is LICOR800CW maleimide, or The first fluorophore is LICOR800CW maleimide and the second fluorophore is 22. The composition of claim 21, wherein the hydroxyl group is LICOR700DX maleimide.

23. The change in the ratio of fluorescence indices is measured at two different wavelengths to determine the concentration of unbound bilirubin. The composition according to any one of claims 21 to 22, which is used for:

24. The emission intensity of the first fluorophore or the second fluorophore is determined based on bilirubin and and hemoglobin, 24. The composition according to any one of claims 23.

25. The bilirubin-responsive probe and / or the non-responsive probe are attached to a solid substrate. The sensor according to any one of claims 1 to 19 or any one of claims 20 to 24, A solid substrate comprising the composition of any one of claims 1 to 4.

26. The solid substrate is Ni-polystyrene, Ni-latex, or Ni-agarose.

26. The solid substrate of claim 25, wherein the solid substrate is a gel.

27. The Ni-polystyrene, Ni-latex, or Ni-agarose beads contain iron. The solid substrate of claim 26 .

28. The bilirubin-responsive probe or the non-responsive probe is selected from the group consisting of SEQ ID NO: 3 7R 16R 20R 29R 37R 46R 50R 88R 92R 94R 10 28. Any of claims 25 to 27, including 0R 125R 129R and 130R (KR14) substitutions. The solid substrate according to any one of claims 1 to 4.

29. The bilirubin-responsive probe and / or the non-responsive probe contain a tag, The solid substrate of any one of claims 25 to 28, wherein the solid substrate comprises a receptor for a tag.

30. The tag is a His-tag, biotin, Flag-epitope, or c-myc epitope. , HA-tag, glutathione-S-transferase (GST), maltose-binding protein Protein (MBP), chitin-binding domain (CBD), thioredoxin, β-galactosidase enzyme, VSV glycoprotein, calmodulin-binding protein, polystyrene (PS) hydrophobic 30. The solid substrate of claim 29, comprising one or more of a hydroxyl group tag, ...

31. wherein the tag is a polyhistidine tag and the solid substrate comprises an immobilized metal chelate.

31. The solid substrate according to any one of claims 29 to 30.

32. The first fluorophore is attached to a cysteine ​​residue on the bilirubin-responsive probe. The solid substrate according to any one of claims 25 to 31,

33. The second fluorophore is attached to a cysteine ​​residue on the non-responsive probe. The solid substrate according to any one of claims 25 to 32.

34. 1. A method of calibrating a bilirubin sensor to measure Kd and Rm, comprising: The sensor according to any one of claims 1 to 19 is used to measure the concentration of bilirubin (BT) in an aqueous solution. mixing with a sample; measuring fluorescence; and a step of determining calibration curve parameters from the measured fluorescence by fitting to equation (1); [Equation 1] where R is the measured fluorescence ratio ((I λ１ / I λ２ ), I λ1 is the background from the sample. is the fluorescence intensity from the first fluorophore at wavelength λ minus the λ band, and I λ２ is the sample is the fluorescence intensity from the second fluorophore at wavelength λ2 minus the background. where Ro is the ratio in the absence of bilirubin, BT is the total bilirubin concentration, and PT is the proton concentration. is the probe concentration, and r is the bilirubin probe fluorophore concentration in the absence of the second fluorophore. Fore I λ2 / I λ1 is the ratio, Kd is the equilibrium dissociation constant of the bilirubin probe, and Rm is the ratio R extrapolated to infinite BT, A method comprising:

35. 1. A method for measuring the concentration of free bilirubin [Bf] in a sample, comprising: Optionally, measuring baseline fluorescence of the sample; applying a sample to the sensor according to any one of claims 1 to 19; measuring the fluorescence of the sample; Optionally, subtracting the baseline fluorescence from the fluorescence of the sample to obtain the measured fluorescence; and determining the concentration of [Bf] from the measured fluorescence; A method comprising:

36. Equation (1) is used to calibrate the sensor, and the following equation (2) is used to determine [Bf]: Used, [Equation 2] where R is the measured fluorescence ratio ((I λ１ / I λ2 ) and I λ1 is the first is the fluorescence intensity from the fluorophore, and I λ2 is from the second fluorophore at wavelength λ where Ro is the fluorescence intensity in the absence of bilirubin, r is the fluorescence intensity in the absence of bilirubin, and r is the fluorescence intensity in the absence of bilirubin. I of the probe in the absence of λ２ / I λ１ is the ratio, Kd is the dissociation constant, and Rm is ∞ 36. The method of claim 35, wherein Bf is the minimum R value at Bf and Rm is the R at bilirubin saturation of the probe. How to post.

37. 3. The method of claim 2, wherein the sample is mixed with one or more carrier macromolecules for the bilirubin.

37. The method according to any one of claims 5 to 36.

38. The one or more carrier macromolecules may be albumin, a lipid-binding protein, a lipid vesicle, or 38. The method of claim 37, comprising a cyclodextrin.

39. 39. The method according to any one of claims 35 to 38, wherein the sensor is attached to a solid support. How to post.

40. The Bf concentration can be measured using a disposable microfluidic device that optionally allows for measurement of undiluted blood samples.

40. The method of any one of claims 35 to 39, wherein the temperature is determined using an in vivo device.

41. 41. The method according to any one of claims 35 to 40, wherein the sample is of human, animal, or plant origin. How to post.

42. The sample is whole blood, plasma, serum, urine, CSF, saliva, gastric juice, interstitial fluid, or lymph. The method according to any one of claims 35 to 41.

43. 3. The method of claim 2, wherein the sample is from a patient receiving an intravenous infusion of an oil emulsion.

43. The method according to any one of claims 5 to 42.

44. the sample is from a patient receiving a drug that displaces bilirubin from albumin; and / or such patients may develop a disease or stress disorder from the injected oil emulsion. Any of claims 35 to 43, which is capable of producing a molecule that displaces bilirubin from albumin from the enzyme. The method according to any one of claims 1 to 4.

45. The sample has not undergone phototherapy, blood transfusion or other therapy to reduce bilirubin levels. The method according to any one of claims 35 to 44, wherein the antibody is derived from a patient.

46. Ro is obtained by photobleaching the sample, thereby obtaining a zero level measurement. The method according to any one of claims 35 to 45.

47. substrate; a lens configured to couple to the substrate and including a sample port for receiving a sample; and Call, Bilirubin-responsive probes, non-responsive probes, and antihemoglobin-immobilized probes a substrate having a peptide; wherein the substrate is exposed to UV light having a wavelength in the range of about 145 nm to about 225 nm. The polymer is treated with light, whereby the phototreated polystyrene polymer chains are transferred to the polymer of the substrate. -Cartridge that connects to the chain.

48. 48. The cartridge of claim 47, wherein the substrate is a polystyrene substrate.

49. The substrate has a dark color configured to reduce the reflected intensity of 660 nm excitation light. A cartridge according to any one of claims 47 to 48, comprising a material.

50. 50. The cartridge according to any one of claims 47 to 49, wherein the lens is an acrylic lens. J.

51. The lens is O ２ 51. The method of claim 47, wherein the method is treated with plasma. Cartridge.

52. Bonding the lens to the polystyrene substrate comprises bonding the lens to the polystyrene substrate to a depth of about 0.1 mm or less.

52. The method according to claim 47, wherein a channel is formed through the cartridge and the cartridge is sealed. The cartridge described.

53. The sample is whole blood, plasma, serum, urine, CSF, saliva, gastric juice, interstitial fluid, or lymph. The cartridge according to any one of claims 47 to 52.

54. The cartridge according to any one of claims 47 to 53, wherein the sample is an undiluted sample.

55. 48. The cartridge configured to measure bilirubin at equilibrium.

55. A cartridge according to any one of items 1 to 54.

56. The cartridge is calibrated with a traceable bilirubin standard.

55. A cartridge according to any one of claims 55.

57. Bilirubin standards are commercially available bilirubin used to calibrate the probes. The probe is used to calibrate the calibration complex, which in turn calibrates the cartridge.

57. The cartridge of claim 56, for use in

58. one or more collection devices for collecting samples from the patient; A sensor according to any one of claims 1 to 19, or one or more in a suitable carrier. A composition according to any one of claims 20 to 24, comprising a probe; and Optionally, a known concentration of unbound biotin below and / or above a medical decision level is one or more reference standards, including Rubin; Kit including:

Images