Single-step method, kit, and system for determining the concentration of unconjugated bilirubin in biological fluids

Abstract

The identification and use of fluorescently labeled proteins that change their fluorescence index upon binding to bilirubin are described. Probes labeled with cysteine ​​or lysine residues, and with two different fluorophores at both cysteine ​​and lysine, are disclosed. These probes are useful for measuring unbound bilirubin levels in liquid samples.

Description

[Technical Field]

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

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

[0003] This application is filed with an Electronic Sequence Listing entitled FFASC077WOSEQLIST.TXT, created on August 24, 2020, which is 40KB in size. The information in the Electronic Sequence Listing is expressly incorporated herein by reference in its entirety.

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

[0005] Bilirubin is a product of hemoglobin turnover, is practically insoluble in water, and is therefore largely associated with albumin in plasma. However, a small fraction of total plasma bilirubin is soluble in the aqueous phase. This unbound, or free, fraction can penetrate the blood-brain barrier and, at elevated levels, is neurotoxic [Ahlfors CE, Wennberg RP, Ostrow JD, and Tiribelli C, Unbound (Free) Bilirubin: An Improved Paradigm for Assessing Neonatal Jaundice, Clin Chem 55:1288-1299, 2009]. Under normal conditions, total serum bilirubin is maintained at low levels by a regulated balance between bilirubin production and excretion. However, in newborns, regulatory mechanisms may not be sufficiently mature to shift the production-excretion balance in favor of accumulation, often resulting in yellow jaundice in approximately 80% of newborns [Maisels MJ and McDonagh AF, Phototherapy for Neonatal Jaundice, N Engl J Med 358: 920-928, 2008; Bhutani VK, Stark AR, Lazzeroni LC, Poland R, Gourley GR, Kazmierczak S, et al. Pre-release testing for severe neonatal hyperbilirubinemia identifies infants requiring phototherapy, J Pediatr 2013;162:477-82]. In most cases, this imbalance may be benign or even beneficial and resolves spontaneously for most newborns [Wennberg RP, Ahlfors CE, Bhutani VK, Johnson LH, and Shapiro SM, Understanding kernicterus: A challenge for improving the management of jaundiced newborns, Pediatrics 117: 474-485, 2006; Gopinathan V, Miller NJ, Milner AD, and Rice-Evans CA. Antioxidant activity of bilirubin and ascorbic acid in neonatal plasma, FEBS Lett 349:197-200, 1994].Unbound bilirubin concentrations can rise to neurotoxic levels, resulting in disorders ranging from reversible hearing loss to the more severe, rarely fatal, neurological sequelae of kernicterus [Ahlfors CE, Wennberg RP, Ostrow JD and Tiribelli C. Unbound (free) bilirubin: an improved paradigm for assessing neonatal jaundice. Clin Chem 55: 1288-1299, 2009].

[0006] Early intervention with phototherapy or exchange transfusion can treat bilirubin-mediated neurotoxicity in neonates [Maisels MJ and McDonagh AF, Phototherapy for Neonatal Jaundice, N Engl J Med 358: 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, Impact of Common Bilirubin Screening on Severe Hyperbilirubinemia and Phototherapy, Pediatrics 124: 1031-1039, 2009]. Intervention guidelines rely primarily on total bilirubin levels, taking into account gestational age and risk factors [Bhutani VK, Johnson L, and Sivieri EM, Predictive Ability of Charge-Time-Specific Serum Bilirubin for Subsequent Significant Hyperbilirubinemia in Healthy Term and Near-Term Infants, Pediatrics 103: 6-14, 1999]. However, basic biochemical and growing clinical evidence predicts that unbound bilirubin will correlate more accurately with bilirubin-mediated neurotoxicity than total bilirubin [Ahlfors CE et al.]. Unbound (Free) Bilirubin: An Improved Paradigm for Assessing Neonatal Jaundice, Clin Chem 55: 1288-1299, 2009; Wennberg RP et al. Intervention guidelines for neonatal hyperbilirubinemia: evidence-based quagmire, Curr Pharm Des 15: 2939-2945, 2009; Ahlfors CE et al., Unbound bilirubin predicts abnormalities in the automatic auditory brainstem response in a diverse neonatal population, J Perinatol 29: 305-309, 2009; Oh W et al., Influence of clinical status on the association of plasma total and unbound bilirubin with death or adverse neurodevelopmental outcome in very low birth weight infants, Acta Paediatr 99: 673-678, 2010].Therefore, unconjugated bilirubin may be superior to total bilirubin for identifying newborns at risk for bilirubin neurotoxicity [Ahlfors CE. Prediction of bilirubin neurotoxicity in jaundiced newborns. Curr Opin Pediatr 22: 129-133, 2010; Watchko JF and Tiribelli C. Bilirubin-induced neurotoxicity mechanisms and management approaches. N Engl J Med 2013; 369: 2021-30].

[0007] Aggressive phototherapy in premature infants is designed to maintain total bilirubin levels below 5 mg / dL [Morris BH et al., Aggressive versus Conservative Phototherapy for Very Low Birth Weight Infants, N Engl J Med 359: 1885-1896, 2008]. Morris et al. found no difference in outcomes (death or neurodevelopmental disability) between patients treated to maintain total bilirubin levels below 5 mg / dL and those treated to maintain total bilirubin levels below 8 mg / dL. A follow-up study found that outcome correlated well with unconjugated bilirubin levels, but not total bilirubin [Oh W et al., The Influence of Clinical Status on the Association of Plasma Total and Unconjugated Bilirubin Levels with Death or Adverse Neurodevelopmental Outcome in Very Low Birth Weight Infants, Acta Paediatr 99: 673-678, 2010]. This suggests that using total bilirubin to determine when to administer phototherapy may be misleading because the total bilirubin level was not conjugated with unconjugated bilirubin, the toxic fraction of bilirubin. Decoupling of total bilirubin and unconjugated bilirubin can result from the presence of molecules that significantly interfere with bilirubin binding to albumin. For example, even if the total bilirubin level is as low as 1 mg / dL, displacement of just 0.2% of the total bilirubin by interfering molecules would result in an unconjugated bilirubin level of 34 nM. This unconjugated bilirubin level exceeds the level considered toxic in term neonates, and much lower unconjugated bilirubin levels are generally considered toxic for premature infants, such as those in the Morris et al. study [Morris BH et al., Aggressive vs. Conservative Phototherapy for Extremely Low Birthweight Infants, N Engl J Med 359: 1885-1896, 2008].

[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 400 and 600 nm, three variants are produced: the photoisomers Z,E-bilirubin IXα and E,Z-bilirubin IXα, and the derivative Z-bilirubin IXα [Jana Jasproval et al., PLoS ONE DOI:10.1371, 2016; J. Jasheprova et al., Neurostimulatory Effects of Bilirubin Photodegradation Products, Scientific Reports (2018) 8:7444]. Currently, the only FDA-approved method for measuring unbound bilirubin levels in serum samples is the Arrows UB analyzer, which is based on measuring the rate of bilirubin oxidation by HRP peroxidase [H Nakamura & Y, Lee, Microdetermination of Unbound Bilirubin in the Serum of Jaundiced Newborns: An Enzymatic Method Using Peroxidase and Glucose Oxidase, Clinica Chimica Acta, 79: 411-417, 1977]. Bilirubin photoproducts can disrupt peroxidase assessment of unbound bilirubin, assuming that the decrease in bilirubin absorbance is due solely to the Z,Z isomer. The absorption spectra of the Z,E and E,Z isomers overlap with the Z,Z absorbance [Antony F. McDonagh et al., Photoisomers: Confounding Factors in Clinical Peroxidase Measurement of Unbound Bilirubin? Pediatrics 2009;123;67-76]. These and other photoisomers are much more soluble but much less toxic than the Z,Z isomer, so peroxidase assessments will falsely report elevated Bf levels. In addition to bilirubin photoproducts, the liver conjugates bilirubin with glucuronic acid, dissolving it in bile and excreting it, where it eventually passes into the feces. Depending on liver health, some of the conjugated bilirubin may leak from the liver into the circulation.Because glucuronide modification increases solubility compared to unconjugated bilirubin, conjugated bilirubin is unbound and often present at concentrations of 2–50 μM, with a reference range of 0–2 μM [Sanjiv Harpavat et al., Persistent Jaundice in Infants and Normal Newborns. Clinical Chemistry 61:2 330–334 (2015)]. These μM concentrations can interfere with measurements of nM (0–100 nM) unbound Z,Z bilirubin concentrations, particularly peroxidase assays, which do not readily distinguish between bound and unbound bilirubin.

[0009] Many drugs and metabolites can bind to albumin, displacing bilirubin from its bound state on albumin, thereby increasing unbound bilirubin levels, regardless of whether total bilirubin levels are elevated [Spear ML et al., Effect of Varying Doses of 15-Hour Fat Infusion on Bilirubin Binding to Albumin, JPEN J Parenter Enteral Nutr 9:144-147, 1985; Amin SB. Effect of Free Fatty Acids on Bilirubin Albumin-Binding Affinity and Unbound Bilirubin in Premature Infants, JPEN J Parenteral Enteral Nutr 34: 414-420, 2010]. A particularly important bilirubin-displacing metabolite is free fatty acid (FFA). FFAs are always present but are maintained at low levels and do not significantly affect healthy, term neonates. However, under stressful conditions, such as those caused by sepsis, FFA concentrations can increase significantly [Nogueira AC et al., Altered plasma free fatty acid concentrations in septic patients are associated with cardiac dysfunction and decreased heart rate variability, Shock 29: 342-348, 2008]. In addition to illness and stress, preterm infants in the NICU can experience extremely elevated FFA concentrations due to parenteral nutrition with oil emulsions such as Intralipid® [Spear M et al., Effect of a 15-hour fat infusion on bilirubin binding to albumin, JPEN J Parenter Enteral Nutr 9: 144-147, 1985; Effect of Amin SB free fatty acids on bilirubin-albumin binding affinity and unbound bilirubin in premature infants, JPEN J Parenteral Enteral Nutr 34: 414-420, 2010]. FFAs bind to albumin with high affinity, similar to bilirubin.Unlike bilirubin, FFAs have multiple affinity binding sites, such that bilirubin displacement becomes significant only when a significant proportion of albumin-binding sites are occupied by FFAs [Spear ML et al., Effect of Varying Doses of 15-Hour Fat Infusion on Albumin, JPEN J Parenter Nutr 9: 144-147, 1985; Amin SB, Effect of Free Fatty Acids on Bilirubin-Albumin Binding Affinity and Unbound Bilirubin in Premature Infants, JPEN J Parenter Nutr 34: 414-420, 2010]. In neonates receiving Intralipid®, the amount of FFAs available to replace bilirubin is not readily predictable, as factors such as gestational age, enzyme activity, and fat accumulation play a role. [Spear ML et al., Effect of 15-hour fat infusion on bilirubin binding to albumin, JPEN J Parenter Enteral Nutr 9: 144-147, 1985; Amin SB, Effect of free fatty acids on bilirubin-albumin binding affinity and unbound bilirubin in premature infants, JPEN J Parenter Enteral Nutr 34: 414-420, 2010]. Monitoring unbound FFA (FFAu) concentrations during lipid infusions has revealed that elevated FFAu levels significantly increase unbound bilirubin to dangerous levels [Hegyi T et al., Total Bilirubin Decoupled Unbound Free Fatty Acids Treated with Phototherapy Ineffective, Neonatology, 2013;104:184-187; Hegyi et al., Effect of Soybean Lipid Infusion on Unbound Free Fatty Acids and Unbound Bilirubin, J Pediatr 2017;184:45-50]. Furthermore, because the unbound levels of these metabolites depend on many patient-specific factors, only direct monitoring of unbound bilirubin during Intralipid® infusion can identify infants at risk for bilirubin neurotoxicity. This is particularly true for bilirubin, since elevated plasma levels of FFA caused by increased Intralipid® concentrations result in elevated unbound bilirubin concentrations without altering total bilirubin concentrations.

[0010] Intracellular lipid-binding proteins (iLBPs) are a family of low-molecular-weight, single-chain polypeptides. There are four recognized subfamilies. Subfamily I includes proteins specific for vitamin A derivatives such as retinoic acid and retinol. Subfamily II includes proteins specific for bile acids, eicosanoids, and heme. Subfamily III includes intestinal fatty acid-binding proteins (FABPs). Subfamily IV includes all other types of fatty acid-binding proteins [Haunerland NH and Spener F, Fatty acid-binding proteins: insights from genetic manipulation, Prog Lipid Res 43: 328-349, 2004] and a FABP that binds bilirubin with low affinity [Di Pietro SM and Santome JA, Isolation, characterization, and binding properties of two rat liver fatty acid-binding protein isoforms, Biochim Biophys Acta 1478: 186-200, 2000]. The entire family is characterized by a common tertiary fold. The ligand-binding properties of different subfamilies overlap considerably. Both subfamily I (Richieri GV et al., Fatty acid-binding proteins from different tissues exhibit distinct patterns of fatty acid interactions, Biochemistry 39: 7197-7204, 2000) and subfamily II wild-type proteins bind fatty acids and their natural ligands. Furthermore, single amino acid substitutions can interconvert the ligand-binding properties of subfamily I and II proteins (Jakoby MG et al., Ligand-protein electrostatic interactions govern the specificity of retinol- and fatty acid-binding proteins, Biochemistry 32: 872-878, 1993). The disclosures of each reference cited herein are expressly incorporated by reference for purposes of the disclosures set forth herein in their entirety. Summary of the Invention

[0011] Described herein are compositions, kits, devices, systems, and methods relating to near-infrared (NIR) Bf sensors with improved fluorophores, lower levels of interference from drugs and metabolites than other methods, disposable cartridges that allow single-step Bf measurement using less than 5 μL of undiluted blood sample, and methods for calibration. As described herein, the probes can include iLBP labeled with a fluorophore. Probes for unbound bilirubin (Bf or UB) are disclosed that change their fluorescence index upon bilirubin binding and can be used to measure the level of unbound bilirubin in a fluid. The fluorescence index can be any measurable quantity, such as wavelength, intensity, polarization, lifetime, or fluorescence. The unbound bilirubin probes disclosed herein do not significantly bind or undergo significant fluorescence changes in the presence of other analytes present in the fluid in which the unbound bilirubin level is determined. The unbound bilirubin probes described herein can be used in the diagnosis and treatment of hyperbilirubinemia and to assess the risk of bilirubin toxicity. A non-responsive probe is also identified that does not bind to bilirubin, does not undergo a change in fluorescence index in the presence of bilirubin, and whose fluorescence is not affected by other analytes commonly present in the fluid in which unconjugated bilirubin levels are measured. Combined, the non-conjugated bilirubin probe with a first fluorophore and the non-responsive probe with a different fluorophore can produce a Bf sensor, which, in the presence of Bf, undergoes a change in the ratio of the fluorescence index from the first fluorophore to the second fluorophore. Together with a cartridge containing a Bf sensor and a dedicated fluorescence reader, the assay is called the UBCheck assay.

[0012] Some embodiments provided herein relate to a sensor for measuring free bilirubin in a sample. In some embodiments, the sensor comprises a bilirubin-responsive probe labeled with a first 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, and the first and second fluorophores fluoresce at different wavelengths. In some embodiments, the bilirubin-responsive probe comprises a first intracellular lipid-binding protein (iLBP), wherein the first iLBP has a peptide sequence comprising SEQ ID NO:1 and includes arginines substituting 14 accessible lysines (KR14 as shown in SEQ ID NO:2); a C-terminal double His-tag linker (C2XH11) having the sequence shown in SEQ ID NO:3; an N-terminal addition of MGI; and up to 62 amino acid substitutions and additions, including a single cysteine. In some embodiments, the bilirubin-responsive probe comprises the sequence of any one of the probes listed in Table 1. In some embodiments, the non-responsive probe comprises a second iLBP, the second iLBP having a peptide sequence comprising SEQ ID NO:1, and including substitutions at positions 72, 73, 74, 126, and 131; a substitution to Cys at any one of positions 27, 31, 33, 54, 73, 74, 76, or 98; up to three additional amino acid substitutions; and a C-terminal double His-tag linker (C2XH11) having the sequence set forth in SEQ ID NO:3. In some embodiments, the non-responsive probe comprises the sequence of any one of the probes set forth in Table 2. In some embodiments, the first fluorophore and the second fluorophore are different fluorophores. In some embodiments, the bilirubin-responsive probe comprises a single cysteine ​​to which a first fluorophore is attached. In some embodiments, the non-responsive probe comprises a single cysteine ​​to which a second fluorophore is attached. In some embodiments, the first fluorophore and the second fluorophore are excited at the same or approximately the same wavelength. In some embodiments, the first fluorophore is LICOR700DX maleimide or LICOR800CW maleimide attached to a cysteine ​​substitution.In some embodiments, the bilirubin-responsive probe is configured to bind to the unconjugated IX-α(Z,Z) isomer of bilirubin. In some embodiments, the bilirubin-responsive probe is configured to minimally bind to bound bilirubin (less than 4 mg / dL). In some embodiments, the bilirubin-responsive probe is configured to not bind to the Z,E or E,Z photoisomers of bilirubin, lumirubin, fatty acids, 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 configured to not bind to the unconjugated IX-α(Z,Z) isomer of bilirubin or bound bilirubin. In some embodiments, the non-responsive probe is configured to not bind to the Z,E or E,Z photoisomers of bilirubin, lumirubin, fatty acids, any other naturally occurring blood components, and / or neonatal drugs. In some embodiments, the first fluorophore is LICOR700DX maleimide and the second fluorophore is LICOR800CW maleimide attached to a cysteine ​​substitution, or when the first fluorophore is LICOR800CW maleimide, the second fluorophore is LICOR700DX maleimide. In some embodiments, the first fluorophore or the second fluorophore is attached to a cysteine ​​substitution, where the cysteine ​​substitution is at position 22, 24, 25, 26, 27, 29, 30, 33, 54, 74, 76, 97, or 98 of SEQ ID NO:1. In some embodiments, the emission intensity of the first fluorophore or the second fluorophore is not affected by the absorbance of a blood component selected from bilirubin and hemoglobin. In some embodiments, the bilirubin-responsive or non-responsive probe further comprises at least one linker.

[0013] Some embodiments provided herein relate to compositions comprising any of the sensors described herein. In some embodiments, the composition comprises a free bilirubin (Bf) sensor. In some embodiments, the sensor comprises a first intracellular lipid-binding protein (iLBP) that binds to bilirubin and is labeled with a first fluorophore, and a second iLBP that does not bind to bilirubin and is labeled with a second fluorophore, wherein the second fluorophore is not bound to the first iLBP, the first fluorophore and the second fluorophore are excited at the same wavelength, the first fluorophore and the second fluorophore have different emission wavelengths, and the second fluorophore does not change its emission in the presence of bilirubin. In some embodiments, the first fluorophore is LICOR700DX maleimide and the second fluorophore is LICOR800CW maleimide, or the first fluorophore is LICOR800CW maleimide and the second fluorophore is LICOR700DX maleimide. In some embodiments, the change in the ratio of the fluorescence indices is measured at two different wavelengths and used to determine the concentration of unbound bilirubin. In some embodiments, the emission intensity of the first fluorophore or the second fluorophore is not affected by the absorbance of a blood component selected from bilirubin and hemoglobin.

[0014] Some embodiments provided herein relate to solid substrates comprising the sensors described herein or the compositions described herein. In some embodiments, the bilirubin-responsive probe and / or the non-responsive probe are attached to a solid substrate. In some embodiments, the solid substrate is Ni-polystyrene, Ni-latex, or Ni-agarose beads. In some embodiments, the Ni-polystyrene, Ni-latex, or Ni-agarose beads contain iron. In some embodiments, the bilirubin-responsive probe or the non-responsive probe contains the substitutions 7R, 16R, 20R, 29R, 37R, 46R, 50R, 88R, 92R, 94R, 100R, 125R, 129R, and / or 130R (KR14) set forth in SEQ ID NO:3. In some embodiments, the bilirubin-responsive probe and / or the non-responsive probe contains a tag, and the solid substrate contains a receptor for the tag. In some embodiments, the tag comprises one or more of a His-tag, biotin, Flag epitope, c-myc epitope, HA-tag, glutathione-S-transferase (GST), maltose-binding protein (MBP), chitin-binding domain (CBD), thioredoxin, β-galactosidase, VSV glycoprotein, calmodulin-binding protein, polystyrene (PS) hydrophobic tag, or metal affinity tag. In some embodiments, the tag is a polyhistidine tag, and the solid substrate comprises an immobilized metal chelate. In some embodiments, a first fluorophore is attached to a cysteine ​​residue on the bilirubin-responsive probe. In some embodiments, a second fluorophore is attached to a cysteine ​​residue on the non-responsive probe.

[0015] Some embodiments provided herein relate to a method of calibrating a bilirubin sensor to determine Kd and Rm. In some embodiments, the method includes mixing any one of the aforementioned sensors with an aqueous sample of known concentration of bilirubin, Bt, measuring fluorescence, and determining calibration parameters from the measured fluorescence by fitting to the following equation (1):

[0016]

number

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

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

[0019]

number

[0020] In some embodiments, equation (1) is used to calibrate the sensor and equation (2) is used to determine [Bf], where R is the measured fluorescence ratio ((I λ1 / I λ2 ) and I λ1is the fluorescence intensity from the first fluorophore at wavelength λ1, and I λ2 is the fluorescence intensity from the second fluorophore at wavelength λ, 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 where Kd is the dissociation constant, Rm is the minimum R value at ∞Bf, and Rm is the R at bilirubin saturation of the probe.

[0021] In some embodiments, the sample is mixed with one or more carrier macromolecules for bilirubin. In some embodiments, the one or more carrier macromolecules include albumin, lipid-binding proteins, lipid vesicles, or cyclodextrins. In some embodiments, the sensor is attached to a solid support. In some embodiments, the Bf concentration is determined using a disposable microfluidics device, which optionally allows for measurement of undiluted blood samples. In some embodiments, the sample is from a human, animal, or plant. In some embodiments, the sample is whole blood, plasma, serum, urine, CSF, saliva, gastric juice, interstitial fluid, or lymph. In some embodiments, the sample is from a patient receiving an intravenous infusion of an oil emulsion. In some embodiments, the sample is from a patient receiving a drug that displaces bilirubin from albumin, and / or such patient may be producing a molecule that displaces bilirubin from albumin from the infused oil emulsion. In some embodiments, the sample is from a patient receiving phototherapy, blood transfusion, or other treatment that reduces bilirubin levels. In some embodiments, Ro is obtained by photobleaching the sample, thereby obtaining a zero level measurement.

[0022] Some embodiments provided herein relate to a cartridge. In some embodiments, the cartridge is configured to measure bilirubin in a sample. In some embodiments, the cartridge includes a substrate; a lens configured to bind to the substrate and including a sample port for receiving a sample; and a substrate having bilirubin-responsive probes, non-responsive probes, and anti-hemoglobin peptides immobilized thereon. In some embodiments, the substrate is treated with UV light having a wavelength ranging from about 145 nm to about 225 nm, thereby linking phototreated polystyrene polymer chains to polymer chains of the substrate. In some embodiments, the substrate is a polystyrene substrate. In some embodiments, the substrate includes a dark-colored material configured to reduce the reflection intensity of 660 nm excitation light. In some embodiments, the lens is an acrylic lens. In some embodiments, the lens is treated with O2 plasma. In some embodiments, bonding the lens to the polystyrene substrate forms a groove having a depth of about 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 lymph. 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, the bilirubin standard is a commercially available bilirubin used to calibrate the probe, the probe is used to calibrate the calibration complex, and the calibration complex is used to calibrate the cartridge.

[0023] Some embodiments provided herein relate to kits. In some embodiments, the kits include one or more collection devices for collecting samples from patients, any one of the sensors described herein, or any one of the compositions described herein, including one or more probes in a suitable carrier, and one or more reference standards containing known concentrations of unbound bilirubin below and / or above a medically critical level. In some embodiments, the 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. In the drawings, like symbols typically identify like components unless context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure, as generally described herein and illustrated in the drawings, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are expressly contemplated herein.

[0026] U.S. Patent Nos. 5,470,714, 6,444,432, 7,601,510, 9,134,317, 9,529,003, and 9,817,004 describe methods for generating probes for determining unbound analytes, and are expressly incorporated herein by reference in their entirety. These probes were constructed using either native or mutant forms of proteins from the intracellular lipid-binding protein (iLBP) family. As mentioned above, this family includes fatty acid-binding proteins (FABPs) [Banaszak et al., Lipid-binding protein A family of fatty acid and retinoid transport proteins, Adv Protein Chem 45:89-151, 1994; Bernlohr DA et al., Intracellular lipid-binding proteins and their genes, Annu Rev Nutr 17:277-303, 1997]. iLBP is an intracellular protein with a molecular weight of approximately 15 kDa, and in the wild-type protein, it has one or two binding sites that bind to FFAs as well as other metabolites.

[0027] Each of the patents and publications disclosed and described herein is expressly incorporated herein by reference in its entirety, and any disclosures specifically referenced herein describe an unbound bilirubin (UB), also referred to herein as a free bilirubin (Bf) ratio sensor, consisting of a bilirubin-sensitive iLBP having one fluorophore and a second fluorophore free or attached to a protein that does not bind or respond to bilirubin. The present disclosure describes improvements over prior patents and publications, including, for example, a near-infrared (NIR) Bf sensor with improved fluorophores, lower levels of interference from drugs and metabolites than other methods, a disposable cartridge that allows for single-step Bf determination in less than 5 μL of undiluted blood sample, and a method for calibration of the assay.

[0028] The bilirubin-sensitive NIR fluorescently labeled iLBP mutants described herein were developed for improved bilirubin specificity and sensitivity. Previous disclosures, such as U.S. Patent No. 9,529,003, describe iLBPs labeled with fluorophores primarily at lysines, terminal amino groups, or cysteines. As disclosed in U.S. Patent No. 9,134,317, labeling at a single cysteine ​​improves sensitivity and specificity for detecting ligand binding to iLBP by eliminating fluorophore labeling at multiple sites (wild-type FABP (including the protein having the sequence shown in SEQ ID NO:1) or mutant FABP proteins (e.g., mutant proteins having the sequences shown in Tables 1 and 2 lacking cysteine ​​residues)). Labeling at most sites reduces the fluorescence signal-to-noise ratio upon ligand binding because fluorophores at most sites do not change their fluorescence upon ligand binding. In addition to the terminal amino group, iLBP has as many as 14 surface-accessible lysines, all of which can be labeled with amino-reactive fluorophores. One improvement of the present disclosure relates to the use of newly available cysteine-specific forms (maleimides) of two fluorophores, LICOR700DX and LICOR800CW. LICOR700DX maleimide was not available or known at the time of the previous disclosure, and LICOR800CW maleimide was not studied in U.S. Patent No. 9,529,003. The bilirubin-sensitive iLBP disclosed herein has a specific single cysteine ​​mutation that is labeled with LICOR700DX-maleimide. An iLBP mutant labeled with a single cysteine ​​mutation with LICOR800CW-maleimide has also been developed that shows no change in fluorescence in the presence of bilirubin.

[0029] The probes disclosed herein may be insensitive to over 50 of the most prescribed drugs for neonates (e.g., those listed in Table 4), conjugated bilirubin, photoisomers of bilirubin, intravenous lipid emulsions used to provide parenteral nutrition to premature or otherwise at-risk infants, and are insensitive to 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 disposable plastic cartridge containing a NIR fluorescent Bf sensor as a dried spot in the center of a blood sample microchannel (e.g., having a volume of 1-50 μL, in some embodiments, 5 μL). Fluorescence from the cartridge can be measured after applying a blood sample by inserting the cartridge into a fluorescence reader specifically developed for this measurement. This configuration of the disposable sample cartridge and reader may allow blood levels of unbound bilirubin to be measured in a single step, in some embodiments using a microliter volume of sample.

[0031] Drying the Bf sensor on the cartridge results in bilirubin-mediated incomplete quenching of the bilirubin-sensitive probe's fluorescence, necessitating new analytical methods for sensor characterization and for calculation of the unbound bilirubin concentration from the ratio of the fluorescence from the bilirubin-sensitive probe divided by the fluorescence from the bilirubin-insensitive probe.

[0032] Accordingly, some embodiments provided herein are directed to methods for identifying fluorescently labeled proteins (or iLBP mutant proteins) that are highly specific for unbound bilirubin and fluorescently labeled iLBP mutant proteins that are unresponsive to bilirubin. Some embodiments are directed to the use of different near-infrared (NIR) fluorophores on bilirubin-responsive and unresponsive iLBP mutant proteins. When combined in a Bf sensor, the presence of Bf results in a change in the ratio of the refractive indexes of the two different NIR fluorophores. Embodiments provided herein relate to methods using two fluorescently labeled proteins to create a fluorescence ratio sensor for determining unbound bilirubin concentration in samples ranging from simple aqueous solutions to complex biological samples including human fluids (blood, CSF, urine, interstitial fluid). In some embodiments, the method involves creating a probe using the methods of U.S. Patent Nos. 7,601,510 and 9,529,003, and creating a ratio Bf sensor through the use of a second fluorescently labeled, unresponsive protein. One or both fluorescent proteins may be free in solution, or one or both may be attached to a solid substrate (matrix) or resin polymer, such as polydextran or polystyrene. In some embodiments, the method includes calibrating the sensor to determine the sensor's calibration constant using Equation (1). Some embodiments involve coupling the probe to the solid substrate. Characteristics of the probe coupled to the solid substrate can be tested to assess accurate and precise determination of unbound bilirubin levels in such devices. Such characteristics may include, for example, the effects of probe dissociation, albumin buffering, or bilirubin binding to the polymer, but not the probe to polymer equilibrium rate. In some embodiments, Bf sensor specificity can be refined by testing against a panel of potential interferents, including common metabolites, drugs, bilirubin photoisomers, bound bilirubin, or other analyte contributions corresponding to less than 1 nM unbound bilirubin or some medically relevant level.In some embodiments, the method further comprises testing the quantification by measuring unbound bilirubin in defined human plasma, serum, or whole blood spiked with bilirubin to ensure specificity for unbound bilirubin in human blood samples. In some embodiments, the method further comprises calculating the unbound bilirubin concentration as described in Equation (2).

[0033] Some embodiments are directed to probes based on iLBPs, such as the lipid-binding protein corresponding to SEQ ID NO:1, that contain one or more amino acid substitutions and a fluorophore (see examples in Tables 1 and 2). In some embodiments, the fluorophore is attached to a cysteine ​​residue of an iLBP that has only a single reactive cysteine. In some embodiments, a bilirubin-sensitive iLBP binds to the IXα-Z,Z isomer of bilirubin but does not significantly bind to the photoisomers Z,E, E,Z and lumirubin, conjugated bilirubin, or fatty acids; a non-responsive iLBP also has only a single reactive cysteine ​​and does not bind or respond to any of these analytes.

[0034] In some embodiments, the bilirubin-sensitive probe corresponds to the lipid binding protein of SEQ ID NO:1 having an N-terminal MGI substitution, a C-terminal double HIS tag substitution C2XH11 (SEQ ID NO:3), and one or more amino acid substitutions at positions selected from 14, 18, 23, 28, 24, 25, 26, 27, 29, 30, 33, 38, 54, 60, 73, 74, 76, 97, 98, 106, 115, 117, or 132 of SEQ ID NO:1.

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

[0036] In some embodiments, the probe is substituted with arginine at positions 7R16R20R29R37R46R50R88R92R94R100R125R129R and / or 130R (KR14-SEQ ID NO:2) of SEQ ID NO:1, except that position 29 is mutated to cysteine.

[0037] In some embodiments, the probe comprises a linker (C2XH11) at the C-terminus of SEQ ID NO:1 having the sequence RGAASHHHHHHSHRATPNTSPHHHHHHH (SEQ ID NO:3).

[0038] In some embodiments, the polynucleotide template encodes an iLBP mutein having a cleavable or non-cleavable affinity tag. In some embodiments, the template polynucleotide template encodes an iLBP mutein having a polyhistidine affinity tag, and the solid substrate comprises an immobilized metal chelate.

[0039] In some embodiments, bilirubin-responsive and non-responsive iLBP mutant proteins are labeled with a single fluorophore at a pH below 8. At a pH below 8, the fluorophore can react with cysteine ​​side chains. In some embodiments, the fluorophore is a thiol-specific fluorophore, e.g., LICOR700DX-maleimide and LICOR800CW-maleimide, that excites at a wavelength of about 660 nm and emits at wavelengths of about 700 nm and 819 nm.

[0040] In some embodiments, the second fluorophore is provided by adding a fluorophore to a non-responsive probe, such as a non-responsive iLBP mutein protein. The non-responsive iLBP is also labeled with a single fluorophore that preferentially reacts with cysteine ​​side chains at a pH below 8. In some embodiments, the fluorophore is LICOR800CW-maleimide or BiotiumCF800-maleimide. In some embodiments, the non-responsive iLBP has zero or a significantly reduced response in its fluorescence index upon exposure to bilirubin compared to the fluorescence of a bilirubin-responsive (bilirubin-binding) iLBP mutein probe.

[0041] In some embodiments, the non-responsive probe is based on an iLBP, such as the lipid binding protein corresponding to SEQ ID NO:1, which contains one or more amino acid substitutions and a fluorophore (see example in Table 2). In some embodiments, the fluorophore is attached to a cysteine ​​residue of an iLBP that has only a single reactive cysteine.

[0042] In some embodiments, the non-responsive probe corresponds to the lipid binding protein of SEQ ID NO:1 having one or more amino acid substitutions at positions selected from 14, 18, 20, 23, 27, 29, 33, 54, 72, 73, 74, 76, 98, 100, 117, 126, or 131.

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

[0044] Some embodiments provided herein are directed to compositions having an iLBP mutant protein labeled with a first fluorophore and a second fluorophore bound to a separate, unbound iLBP that does not bind to bilirubin. In some embodiments, the first and second fluorophores can be excited at the same wavelength, and the emission wavelengths of the first and second fluorophores are different. In some embodiments, the second fluorophore is unaffected (does not change its emission) in response to bilirubin binding to the bilirubin-unresponsive iLBP mutant protein, and / or the unresponsive iLBP does not bind to bilirubin. In some embodiments, the first fluorophore is LICOR700DX maleimide and the second fluorophore is LICOR800CW maleimide.

[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 the concentration of unbound bilirubin.

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

[0047] In some embodiments, the first fluorophore is attached to a cysteine ​​and is LICOR700DX-maleimide, and the second fluorophore is also attached to a cysteine ​​on a different iLBP and is LICOR800CW-maleimide.

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

[0049] In other embodiments described herein, a second, different fluorophore is attached to a protein that does not bind bilirubin. A probe conjugated with a first fluorophore attached to a bilirubin-responsive iLBP mutant protein and a probe conjugated with a second fluorophore binds to a bilirubin-unresponsive iLBP mutant protein. Thus, a mixture of the responsive and unresponsive probes produces a Bf sensor that changes the ratio of fluorescence indices measured at two different wavelengths in response to bilirubin. The second fluorophore can have a longer or shorter emission wavelength than the first (responsive iLBP mutant protein) fluorophore, but both fluorophores should share a common excitation wavelength. For example, in some embodiments, the first (protein-bound) fluorophore is LICOR700DX maleimide, and examples of the second include, but are not limited to, LICOR800CW maleimide and / or BiotiumCF800 maleimide attached to a bilirubin-unresponsive protein. One or both fluorescently labeled proteins may be free in solution or embedded in another polymer or solid substrate. This arrangement has the desirable advantage that the concentrations of the fluorophores can be adjusted so that the emission intensities of both fluorophores are similar, even when the excitation maximum of the second fluorophore is different from that of the first fluorophore. This type of ratio probe, which uses a second, different fluorophore that is not bound to the same protein as the first fluorophore, eliminates the problem of energy transfer quenching of one fluorophore by the other, which typically occurs when both fluorophores are located on the same protein.

[0050] In some embodiments, the second fluorophore is attached to an acceptor protein. In some embodiments, the probe contains substitutions 7R16R20R29R37R46R50R88R92R94R100R125R129R and 130R (KR14-SEQ ID NO:2) of SEQ ID NO:1, except that one of these positions has a cysteine ​​substitution.

[0051] In some embodiments, the probe comprises a C-terminal linker C2XH11 composed of the sequence RGAASHHHHHHSHRATPNTSPHHHHHHH (SEQ ID NO:3).

[0052] Embodiments provided herein relate to probes in which the fluorophore is attached to a cysteine ​​residue (e.g., LICOR700DX maleimide, LICOR800CW maleimide, LICOR, IRDye680LT maleimide, Alexafluor680 maleimide, or BiotiumCF800 maleimide).

[0053] In some embodiments, any of the above probes may contain two or more tags at the C-terminus or N-terminus of the probe in combination with one or more linkers for attachment to a solid support.

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

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

[0056] Some embodiments provided herein relate to bilirubin-sensitive probes (e.g., LICOR700DX-maleimide) and bilirubin-unresponsive probes (e.g., LICOR800CW-maleimide) free in solution. In some embodiments, the bilirubin-sensitive probes and bilirubin-unresponsive probes are attached to a solid substrate in solution.

[0057] The embodiments provided herein are directed to solid substrates comprising any of the above-mentioned probes attached to the solid substrate. In some embodiments, the solid substrate is a polystyrene or latex bead, Ni-polystyrene bead, optionally containing iron. The probe selected for attachment to the solid substrate can contain any of the above modifications alone or in combination, including, but not limited to, N-terminal and C-terminal modifications, linkers, and surface lysine (KR14) substitutions. Two probes with different emission wavelengths can be immobilized on the same or different solid substrates, such as nanoparticles or beads.

[0058] In some embodiments, the probes are tagged for attachment to a solid substrate, in some embodiments, the tag comprises one or more of a His tag, biotin, a Flag epitope, a c-myc epitope, an HA tag, glutathione-S-transferase (GST), maltose binding protein (MBP), a chitin-binding domain (CBD), thioredoxin, β-galactosidase, VSV-glycoprotein, calmodulin-binding protein, a polystyrene (PS) hydrophobic tag, or a metal affinity tag.

[0059] Some embodiments are directed to solid substrates in which the probe has a tag and the solid substrate comprises a receptor for the tag, in some embodiments the tag is a polyhistidine tag with or without an additional linker, and the solid substrate comprises an immobilized metal chelate.

[0060] Other embodiments include bilirubin probes attached to nanoparticle solid substrates, including, but not limited to, polymers such as dextran, polystyrene, latex, agarose beads, or Ni-NTA polystyrene beads (optionally containing iron). These nanoparticle substrates can be further immobilized on macroscopic surfaces. Examples of the use of such surfaces include, but are not limited to, grooves in disposable microfluidic devices (including single-use sample cartridges). Examples of bilirubin probes designed to be immobilized on surfaces include, but are not limited to, combinations of each from Tables 1 and 2, whose dual His tags and linkers allow for chelation with various metal ligands on various polymer resins, including, but not limited to, Ni, Co, or Cu on polystyrene, latex, or agarose beads. Such probe-attached beads can be used freely in solution, and bilirubin-responsive and non-responsive probes can be added to the same or different beads. For both configurations, the two different fluorophores are sufficiently separated to eliminate energy transfer and thereby obtain a relative response to bilirubin binding. In some embodiments, the substrate is a microfluidic device or a multiwell plate. In some embodiments, the substrate is included in a detection device described herein such that it is attached to a surface of the cartridge.

[0061] Embodiments provided herein are directed to iLBP muteins having a single cysteine ​​labeled with a fluorescent dye. In some embodiments, any surface lysine or any other cysteine ​​that has fluorescent labeling activity under cysteine / lysine-specific labeling conditions is replaced with another amino acid, including, for example, alanine or arginine. In some embodiments utilizing an iLBP mutein template corresponding to SEQ ID NO:1, the lysine at position 27 is highly reactive and can typically be mutated to alanine unless a label is directed to that position.

[0062] In some embodiments, bilirubin is complexed with a carrier polymer, such as albumin, a lipid-binding protein, a lipid vesicle, or a cyclodextrin. The complex of bilirubin with the carrier polymer buffers the concentration of unbound bilirubin, providing clamping of the level of unbound bilirubin. In some embodiments, the carrier polymer is albumin. In further embodiments, the albumin is, for example, human serum albumin (HSA), which has a greater affinity for bilirubin than bovine serum albumin and therefore, in some embodiments, may be a more preferred albumin buffer for bilirubin.

[0063] Embodiments provided herein are directed to methods of calibrating a bilirubin probe by mixing the sensor with samples of bilirubin of increasing bilirubin concentrations (BT) in an aqueous medium, measuring the ratio of the fluorescence intensities R of the sensor at each concentration, and determining the calibration parameters (Kd, Rm, and R) from the measured fluorescence by fitting to equation (1), where R is the measured fluorescence ratio (I λ1 / I λ2 ), where I λ1 is the fluorescence intensity from the first fluorophore at wavelength λ1, and I λ2 is the fluorescence intensity from the second fluorophore at wavelength λ, and λ and I λ2 where both are background subtracted, Ro is the ratio in the absence of bilirubin, BT is the total bilirubin concentration, PT is the concentration of the sensor, and r is the I of the fluorescence of the bilirubin-sensitive probe in the absence of the second fluorophore. λ2 / I λ1 where Kd is the equilibrium dissociation constant of the sensor, and Rm is the ratio R extrapolated to infinite BT. Equation (1) is used in some embodiments when Rm > 0, rather than equation (5) of U.S. Pat. No. 9,529,003.

[0064] Embodiments provided herein are directed to methods for measuring the concentration of free bilirubin [Bf] by a combination of the following steps, including optionally measuring the fluorescence of a sample, mixing a sensor with the sample, measuring the fluorescence, and optionally subtracting the fluorescence intensity of the sample in the absence of the sensor (background or blank) from the sample fluorescence intensity in the presence of the sensor, calculating R from the background-subtracted sensor intensities, and determining the concentration of [Bf] from equation (2).

[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 fluorescence intensity from the first fluorophore at wavelength λ1, and I λ2 is the fluorescence intensity from the second fluorophore at wavelength λ2, both intensities subtracted by the sample blank, Ro is the ratio in the absence of bilirubin, and r is the I of the sensor in the absence of the second fluorophore. λ2 / I λ1 where Kd is the dissociation constant, Rm is the minimum R value at ∞Bf, and Rm is the R at bilirubin saturation of the probe.

[0066] In some embodiments, the sensor is comprised of two fluorophores, with one fluorophore in combination with a bilirubin-responsive iLBP, and the second fluorophore is attached to a polymer or protein free in solution that does not bind to bilirubin. In some embodiments, the sensor is comprised of a protein with one fluorophore that binds or responds to bilirubin and is attached to a solid substrate, and the second fluorophore is attached to another protein that does not bind or respond to bilirubin, also attached to a solid substrate, but that is separated from the protein with the first fluorophore.

[0067] In some embodiments, the sample comprises a carrier macromolecule for bilirubin, such as albumin, a lipid binding protein, a lipid vesicle, or a cyclodextrin.

[0068] In some embodiments, bilirubin-responsive and non-responsive probes are attached to the channels of a disposable microfluidic channel, allowing for the measurement of Bf in undiluted blood samples.

[0069] In some embodiments, the sample is from a human, animal, or plant. In some embodiments, the sample is from whole blood, plasma, serum, urine, CSF, saliva, gastric juice, interstitial fluid, or lymph. In some embodiments, the sample is from a patient receiving an intravenous infusion of an oil emulsion. In some embodiments, the sample is from a patient who, due to disease or stress, may produce molecules that displace bilirubin from albumin. In some embodiments, the sample is from a patient being treated with a drug that can displace bilirubin from albumin. In some embodiments, the sample is from a patient receiving phototherapy, blood transfusion, or other treatment that reduces bilirubin levels.

[0070] Embodiments provided herein are directed to kits that may include one or more collection devices for collecting samples from a patient, one or more sensors as described above, or a composition comprising one or more sensors in a suitable carrier, and, optionally, a reference standard comprising a known concentration of unbound bilirubin.

[0071] The embodiments provided herein are directed to probes defined in either Table 1 or 2.

[0072] For purposes of this disclosure, bilirubin is the Z,Z isomer of unconjugated bilirubin IXα [McDonagh AF et al., Photoisomers: Confounders in Clinical Peroxidase Measurements of Unconjugated Bilirubin? Pediatrics 123: 67-76, 2009]. Unconjugated bilirubin is the aqueous monomer of unconjugated Z,Z IXα bilirubin, which is distinct from the bilirubin typically found bound to albumin in plasma.

[0073] For purposes of this disclosure, the term "lipid" is taken to have its ordinary accustomed meaning and defines a compound that is most soluble in organic solvents but has some level of solubility in the aqueous phase (unbound fraction). Thus, "lipid-binding protein" includes any protein that can bind to lipids, as lipids are defined herein.

[0074] Levels of unbound molecules, such as bilirubin, lipids including fatty acids, hormones, and metabolites, can provide diagnostic information for health and disease when measured in appropriate human or animal biological fluids. It is becoming increasingly clear that determining the unbound (also referred to herein as "aqueous-phase" or "free") concentrations of such molecules provides important information regarding physiological homeostasis. Many metabolites are hydrophobic molecules with poor water solubility, have unbound concentrations much lower than their "total" concentrations, and the majority of the "total" concentration can be bound to proteins or cells. In biological fluids, the concentration of unbound molecules is often regulated to maintain a relatively constant unbound concentration under normal physiological conditions. This regulation occurs through interactions between the molecules and carrier proteins, such as albumin. Thus, the majority of molecules are generally bound to albumin or other carriers. However, a small amount of molecules can dissociate (and rebind) from albumin into the aqueous phase; these are unbound molecules.

[0075] For purposes of this disclosure, in some embodiments, a "bilirubin sensor" refers to two iLBPs labeled with two different fluorophores at cysteine ​​residues, where the first iLBP undergoes a change in fluorescence index upon binding to bilirubin, and the second iLBP does not significantly change its fluorescence in the presence of bilirubin (a non-responsive probe). In some embodiments, the bilirubin sensor may also include an iLBP fluorescently labeled at cysteine ​​residues, with additional fluorescence provided by a second fluorophore that is free in solution and attached to a different molecule or polymer, such that the second fluorophore does not change fluorescence in the presence of bilirubin. In this case, if the fluorescence of only one of the fluorophores changes upon binding to bilirubin, the ratio of the fluorescence indices at the two wavelengths will be different. Such probes can be used to specifically determine the aqueous concentration of unbound bilirubin, which is otherwise difficult due to its poor solubility in aqueous solutions and the presence of other metabolites, particularly free fatty acids. The change in the ratio of fluorescence response is particularly important for the accurate determination of intracellular concentrations of unbound bilirubin, and is important for improving the accuracy and precision of the determination of extracellular concentrations of unbound bilirubin.

[0076] U.S. Patent Nos. 7,601,510, 9,134,317, and 9,529,003 (Huber AH et al., "Fatty Acid-Specific Fluorescent Probes and Their Use in Separating a Mixture of Different Unbound Free Fatty Acids in Equilibrium with Albumin," Biochemistry, 45:14263-14274, 2006) and (Huber AH and Kleinfeld AM, "Unbound Free Fatty Acid Profiles in Human Plasma and the Unexpected Absence of Unbound Palmitoleic Acid," J. Lipid Res., 58:578-585, 2017) describe methods for the high-throughput generation of highly specific probes that enable the determination of unbound analytes. U.S. Patent Nos. 7,601,510, 9,134,317, and 9,529,003 describe the development of bilirubin-specific probes. The embodiments provided herein relate to improvements to the bilirubin technology described in U.S. Patent Nos. 7,601,510, 9,134,317, and 9,529,003. The disclosed embodiments improve the accuracy and precision for determining unbound bilirubin levels and allow this technology to be used in different measurement formats. The bilirubin probes described in U.S. Patent Nos. 7,601,510, 9,134,317, and 9,529,003 (Huber et al., "Fluorescent Sensor for Quantifying Unbound 45: Bilirubin Concentration," Clin Chem 58: 869-876, 2012) primarily use the probe in aqueous suspension (cuvette-based fluorescence measurement) and primarily use diluted plasma or serum samples. The embodiments disclosed herein relate to a method for producing a Bf sensor by using two probes: an iLBP mutant protein labeled with a fluorophore that fluoresces in the near-infrared. The first iLBP is labeled with LICOR700DX-maleimide, which fluoresces at 700 nm and whose fluorescence is quenched upon binding to Z,Z bilirubin. The second iLBP is labeled with LICOR800CW-maleimide, which fluoresces at 819 nm and is unresponsive to bilirubin. Both LICOR700DX-maleimide and LICOR800CW-maleimide can be excited at 660 nm. Some embodiments provided herein relate to the quenching of long-wavelength probes by bilirubin.Additionally, some embodiments relate to methods for attaching bilirubin probes to solid surfaces and methods for using such compositions to measure unbound bilirubin in microfluidic devices and disposable sample cartridges.

[0077] Bilirubin sensors are used to determine unbound bilirubin levels in blood samples, and fatty acids are the most abundant metabolites in blood with properties similar to bilirubin. Fatty acids compete with bilirubin for binding to albumin, resulting in unbound concentrations similar to those of unbound bilirubin. Bilirubin-sensitive probes are generally developed starting from iLBP mutants with high affinity for fatty acids. Therefore, the first step in discovering bilirubin probes from iLBP mutant protein probes is to screen over 300,000 such probes using up to 11 of the most abundant fatty acids to identify probes that do not significantly respond to fatty acids. Generally, mutant proteins without fluorescent labels do not elicit a measurable signal upon analyte binding, making systematic high-throughput screening of mutant proteins impossible. The finding that ΔR / ΔRADIFAB2 < 0.1 led to the identification of over 10,000 such fatty acid non-responders ("non-responder library"). This quantitative benchmarking indicates that the affinity of these probes for fatty acids is generally at least 10-fold less than that of the ADIFAB2 reference probe. Screening these non-responsive probes with bilirubin allows further mutagenesis of the newly identified template proteins to identify potential bilirubin probes and / or templates for use in generating new mutein probe libraries. The resulting libraries are screened for response to fatty acids and bilirubin, and the probe identified as most responsive to bilirubin and least responsive to fatty acids can be identified as a bilirubin probe or used as a template for further rounds of mutagenesis and screening. This is performed for all of the bilirubin and non-responsive muteins described herein (e.g., Tables 1 and 2), and the resulting bilirubin sensors have no significant response to fatty acids.

[0078] Bilirubin probes identified by these methods as having useful properties, including significant bilirubin response and low to zero response to fatty acids, are further characterized. Probes that do not significantly respond to FFAs, meaning they bind FFAs, are 10-fold less than they bind to bilirubin. In some embodiments, binding to FFAs is 100-fold less than binding to bilirubin. This involves calibration to determine the probe's bilirubin binding affinity and fluorescence properties, as well as monitoring unbound bilirubin levels in an aqueous solution containing bilirubin and human serum albumin to identify potential competition with fatty acids. Non-responsive probes may be generated 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 may compete with bilirubin for binding to the probe, thereby resulting in inaccurate determination of unbound bilirubin levels. Competition with fatty acids is assessed by determining whether the fluorescence response of the bilirubin probe plus bilirubin changes with the addition of fatty acids.

[0079] Bilirubin probes found by the methods described herein that yield accurate bilirubin concentrations in solutions containing bilirubin and albumin and exhibit no detectable fatty acid competition are selected for further testing in human blood samples. Plasma samples from individual newborn and adult donors, as well as pooled samples from commercial sources, are used to determine whether the bilirubin probes provide accurate serum or plasma unbound bilirubin concentrations in samples with essentially unknown levels of non-bilirubin analytes commonly present in human blood samples. Healthy adults have low bilirubin levels, with bilirubin:HSA molar ratios below 0.1, and therefore, their Bf concentrations approach zero (<1 nM). Blood samples are spiked with bilirubin and albumin concentrations are measured to obtain unambiguous bilirubin:albumin ratios that yield Bf levels above the limit of detection (LOD) of the assay, which is less than 1 nM. The concentration of unbound bilirubin in various samples is then measured with the Bf sensor, and the results are compared with a peroxidase assay [Jacobsen J and Wennberg, RP: Quantitation of Unbound Bilirubin in Neonatal Serum, Clin Chem 20: 783, 1974]. The only FDA-cleared test for Bf was performed using the Arrows UB-2 analyzer [Nakamura H and Lee Y, Microquantitation of Unbound Bilirubin in Jaundice Neonatal Serum: An Enzymatic Method Employing Peroxidase and Glucose Oxidase, Clinica Chimica Acta, 79 (1977) 411-417]. The equivalence of plasma unbound bilirubin concentrations measured by the Bf sensor to those measured by the peroxidase assay confirms that blood components other than unbound bilirubin have no detectable effect on probe performance.

[0080] U.S. Patent Nos. 7,601,510, 9,134,317, and 9,529,003 omit the time-consuming and necessary step of characterizing bilirubin binding to the protein; only the probe itself is characterized. This is necessary not only to avoid characterizing the protein, but also because the properties of the probe are often unpredictable from the ligand-protein binding properties. For example, different proteins may have very similar binding affinities, but the fluorescence response of their derivative probes may vary.

[0081] Most previously described bilirubin probes were labeled solely with acrylodan, primarily at lysine 27 of SEQ ID NO:1 (including U.S. Patent Nos. 7,601,510, 9,134,317, and 9,529,003, and [Huber et al., Fluorescent Sensor for Quantification of Unbound Bilirubin Concentration, Clin Chem 58:869-876, 2012]). Additional bilirubin probes were labeled with two different fluorophores: acrylodan at lysine 27 of SEQ ID NO:1 and Texas Red maleimide at the N-terminal cysteine ​​adduct, in two versions: one without KR14 and the other without KR14 ("KR14" is an abbreviation referring to the following 14 surface lysine-to-arginine mutations in SEQ ID NO:1, including 7R16R20R29R37R46R50R88R92R94R100R125R129R and 130R, with substitutions as shown in SEQ ID NO:2, resulting in reduced multiple acrylodan labeling). These probes had good affinity and response to bilirubin and were not significantly affected by non-bilirubin metabolites in human blood samples. However, due to the presence of high bilirubin concentrations, severe neonatal hyperbilirubinemia [Bhutani VK and Johnson L, Jaundiced Newborns in the Emergency Department: Prevention of Kernicterus, Clin Ped Emerg Med 9:149-159, 2008], and hemoglobin in blood samples, acrylodan-only probes may adversely affect the bilirubin-mediated inner filter effect. Dual-labeled probes containing acrylodan and a longer-wavelength fluorophore (e.g., Texas Red) significantly reduce acrylodan fluorescence intensity due to energy transfer between the acrylodan and the secondary fluorophore, making such probes inaccurate for Bf values ​​in the clinically relevant concentration range.

[0082] To overcome these drawbacks, embodiments provided herein relate to sensors and methods for determining unbound bilirubin levels. A mutein library was identified in which bilirubin-quenched fluorophores labeled a single cysteine ​​side chain, and the location of this side chain was found to be important for optimizing the fluorescence change upon bilirubin binding. Bilirubin probes were also described in which bilirubin-quenched fluorophores labeled different cysteine ​​side chains, and the location of the side chain was found to be important for optimizing the fluorescence change upon bilirubin binding. Also described are bilirubin-quenching fluorophores that absorb and emit at long wavelengths where bilirubin quenching by Förster energy transfer should not occur. This disclosure also relates to the fluorescence quenching by bilirubin of very long-wavelength fluorophores, including those that extend into the infrared. Due to their long-wavelength absorbance and fluorescence, such fluorophores are not affected by bilirubin or hemoglobin absorbance, or the absorbance of virtually any other chromophores potentially present in blood samples.

[0083] Further embodiments described herein relate to methods for producing bilirubin ratio sensors that use a single fluorophore on a bilirubin-binding protein whose fluorescence decreases upon binding to bilirubin and a second, different fluorophore attached to a protein (non-responsive probe) that does not respond to and / or bind to bilirubin. Such sensors respond to bilirubin binding to the protein portion of the probe with a change in the ratio of fluorescence indices measured at two different wavelengths. This type of ratio sensor, which uses a separate second fluorophore, eliminates the problem of energy transfer quenching between fluorophores that is typically observed when both fluorophores are located on the same macromolecule, such as a protein. Such quenching significantly reduces signal intensity, thereby reducing the accuracy and precision of measurements of unbound bilirubin concentration. Avoidance of this energy transfer is achieved by not attaching both fluorophores to the same probe molecule.

[0084] Also described are bilirubin probes that can be attached to solid substrates, such as polystyrene or latex beads, and beads that can be immobilized on surfaces for use in disposable microfluidic devices. First and second fluorophores can be attached to bilirubin-responsive iLBPs and bilirubin-unresponsive iLBPs, respectively. In some embodiments, both probes can be attached to the same or different solid bead substrates, and these beads can be immobilized on a disposable plastic microfluidic device, so that the two fluorophores remain sufficiently separated so that energy transfer is insignificant. A mixture of sensor-bead complexes in an aqueous buffer solution (slurry) is dispensed into a groove in a microfluidic device in a volume of less than 2 μl and dried to form a circular spot approximately 2 mm in diameter, which adheres to the bottom of the groove. An undiluted blood sample is applied to the inlet of the groove and rapidly flows across the dried bilirubin sensor spot, allowing it to reconstitute. The blood sample-containing device is then placed in a fluorescence reader, which measures the ratio of fluorescence from the bilirubin-responsive and non-responsive probes (sensors), from which the Bf concentration is calculated.

[0085] Also described is a method for calibrating and using the bilirubin sensors described herein so that Bf concentrations can be determined in approximately 5 μl of undiluted blood samples in a single step. These small blood samples are applied to disposable plastic microfluidic cartridges containing dried bilirubin ratio sensors. The bilirubin sensor containing the cartridges is preferably used for a single measurement, and calibration parameters (Kd, Rm, Ro) must be determined within a "lot" of identically manufactured cartridges. Calibration is performed by titrating a sufficient number of cartridges with aqueous samples containing increasing levels of distinct Bf. Because the plastic binds unbound bilirubin and the surface-to-volume ratio within the microfluidic channel is large, the calibration Bf samples must be highly buffered by complexing with albumin. The Bf concentration of each complex is determined by measuring each complex by cuvette fluorometry using a free calibrated sensor in aqueous solution. The free sensor is calibrated by titration with aqueous solutions of bilirubin, and the concentration of these unbound bilirubin solutions is determined by absorbance. The free probe response (R value vs. Bf concentration) is used to determine the free sensor calibration (Kd, Rm, Ro) (Equation 1). Using the calibrated free sensor mixed with each BR:HSA complex sample, the Bf concentration of each complex is determined by cuvette fluorimetry using Equation 2. These calibrated complexes are used to determine the binding parameters of the disposable cartridge, including the equilibrium dissociation constant (Kd), minimum fluorescence ratio (Rm), and initial ratio (Ro), under defined conditions of temperature, pH, and solution composition appropriate for blood samples. Binding isotherms are performed in aqueous buffer by measuring the change in fluorescence of the bilirubin sensor in response to increasing bilirubin concentrations ("titration data"). The set of fluorescence responses at each bilirubin concentration is fitted to an appropriate equation ("calibration equation (3)") that accurately describes the fluorescence response as a function of Bf concentration, specific spectral properties, and Kd.

[0086]

number

[0087] The free bilirubin concentration ([Bf]) is determined in samples where [Bf] is buffered by the albumin binding equilibrium and therefore not significantly perturbed by the presence of the bilirubin probe. Equation (2) is used to determine [Bf] for ratio sensors where Rm is >0.

[0088] Some embodiments provided herein relate to the development of fluorescent protein molecules that can be used to determine the concentration of unbound analytes.More specifically, some embodiments relate to: 1) the identification of bilirubin probes produced by the methods of U.S. Patent Nos. 7,601,510, 9,134,317 and 9,529,003, which are expressly incorporated herein by reference, and modifications of these methods are also described; 2) the use of such probes for clinical medicine and basic science; or 3) the example of these probes for determining the concentration of unbound bilirubin in different fluids.

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

[0090] In some embodiments, two different fluorophores are used, one of which labels iLBPs that respond to bilirubin binding and exhibit a change in fluorescence index upon bilirubin binding to the probe. The second fluorophore labels iLBPs that do not respond to or bind to bilirubin. The second probe provides a reference point so that the difference in the ratio of fluorescence at two different wavelengths can be observed upon bilirubin binding. The second probe may not respond to bilirubin binding or may respond in a manner different from the first fluorophore. In some embodiments, the second fluorophore has an emission point at a different wavelength than the first fluorophore. Examples of chemical dyes that can be used as the second fluorophore include, but are not limited to, LI-COR800CW maleimide, Cy7 maleimide, Cy7.5 maleimide, VivoTag-S750-M, and Alexa Fluor 750. In some embodiments, the second fluorophore is LI-COR800CW maleimide.

[0091] In some embodiments, two different fluorophores are used, one of which is conjugated to a cysteine ​​and is responsive to bilirubin binding, i.e., demonstrates a change in fluorescence index upon bilirubin binding to the fluorescently labeled iLBP mutein. The second fluorophore is not chemically conjugated to the bilirubin-binding iLBP mutein and is not sensitive to bilirubin binding to the iLBP mutein. The second fluorophore provides a reference point so that the difference in the ratio of fluorescence at two different wavelengths upon bilirubin binding can be observed. In one embodiment, the second fluorophore has an emission point at a longer wavelength than the first fluorophore. Examples of chemical dyes that can be used as the first fluorophore include, but are not limited to, LI-COR700DX maleimide, Biotum CF680-M, CF680R-M, Lumiprobe Cy5, Cy7, Perkin Elmer Vivotag 645-M, Vivotag 680XL-M, Atto Tek Atto680, Atto700, Dyomics DY677, or DY689. Examples of chemical dyes that can be used as the second fluorophore include, but are not limited to, LI-COR800CW maleimide. In some embodiments, the first fluorophore is LI-COR700DX maleimide and the second fluorophore is LI-COR800CW maleimide.

[0092] iLBP mutant proteins can be "tagged" to bind to solid supports with high affinity. This includes, but is not limited to, tagging with biotin, Flag epitope, c-myc epitope, HA tag, glutathione-S-transferase (GST), maltose-binding protein (MBP), chitin-binding domain (CBD), thioredoxin, β-galactosidase, VSV-glycoprotein, calmodulin-binding protein, polystyrene (PS) hydrophobic tag, or metal affinity tags such as 6XHis tag. The specific association of affinity tags with solid support materials facilitates unbound bilirubin measurement in flat surface configurations, including, but not limited to, multiwell plates and microfluidic devices. Due to their attachment to the solid support, the probes can be concentrated into a limited, effectively two-dimensional area. This allows for measurement of unbound bilirubin within a thin layer of sample solution flowing across the probe, which is limited to the effective two-dimensional area. This effectively enables front-face fluorescence measurement, which reduces absorbance by bilirubin and hemoglobin and facilitates measurement in whole blood. Affinity tags can be fused to either the NH2-terminus or COOH-terminus, or both termini simultaneously, as shown in Tables 1 and 2. In some embodiments, a 6X histidine tag was fused to either the NH2-terminus or COOH-terminus, or both termini simultaneously, of an iLBP mutein without significantly altering the bilirubin-binding properties of the protein. In some embodiments, the fusion peptide consists of two separated histidine regions at the COOH-terminus of the probe. In some embodiments, the probe is immobilized on a solid support, including, but not limited to, Ni-polystyrene beads.

[0093] In some embodiments, the bilirubin sensor immobilized on a solid support combines two probes labeled with different fluorophores that excite at the same wavelength but emit at two different wavelengths. One of the probes is responsive to bilirubin binding, i.e., it exhibits a change in fluorescence index upon bilirubin binding to the protein. The second probe, labeled with a different fluorophore, either does not respond to bilirubin binding or changes in response to bilirubin differently from the first probe. The fluorophore of the second probe provides a reference point so that the difference in the ratio of fluorescence at the two different wavelengths upon bilirubin binding can be observed. In some embodiments, the first bilirubin-sensitive protein is labeled with LI-COR700DX maleimide, and the second bilirubin-insensitive protein is labeled with LI-COR800CW maleimide.

[0094] Some embodiments provided herein relate to disposable sample cartridges containing Bf sensors, as shown in Figures 10A-10D, 11A-11D, and 12. After adding a sample, the cartridge is placed in a fluorescence reader to measure the Bf concentration of the sample. In some embodiments, the cartridge is composed of a polystyrene base and an acrylic lens, as in Figures 10A-10D and 11A-11D. In some embodiments, the sensor is composed of a bilirubin-sensitive iLBP from Table 1, LICOR700DX-maleimide, and a non-responsive iLBP, LICOR800CW-maleimide, from Table 2, and the two probes are bound to Ni-NTA polystyrene beads (e.g., Dynal 1 μm NTA beads) either on separate beads or on the same bead. In some embodiments, a peptide from Table 4 is added to the Ni-NTA polystyrene beads with the bound probes to help reduce interference from hemoglobin and / or hemolysates. Probe and peptide-labeled beads are suspended in an aqueous buffer to form a slurry, which is dispensed onto a polystyrene substrate as droplets of 250–2000 nL volume. This spot of Bf sensor beads is then dried and then encapsulated by an acrylic lens, forming a channel containing the dried sensor spot. In some embodiments, the polystyrene substrate is treated with UV irradiation before applying the sensor slurry. In some embodiments, UV irradiation at a wavelength of approximately 185 nm is superior to 254 nm irradiation for immobilizing the polystyrene bead sensors and forming a well-defined circular sensor spot on the polystyrene substrate. In some embodiments, after the sensor spot is dried, a plasma-treated acrylic lens is snapped onto the polystyrene substrate, forming a sealing groove 2.7 mm wide, 0.1 mm high, and 13.7 mm long (total volume = 3.7 μl). A sample is then applied to the sample port of the lens, which rapidly fills the channel and reconstitutes the dried sensor.

[0095] In some embodiments, the cartridge is a microfluidic device and includes a polystyrene substrate, an acrylic lens, and a substrate having bilirubin-responsive probes, non-responsive probes, and an antihemoglobin peptide immobilized thereon. In some embodiments, the polystyrene substrate includes a material having a dark color configured to reduce the reflection intensity of 660 nm excitation light, such as a dark gray substrate. In some embodiments, the substrate is beads to which the probes and / or antihemoglobin peptides are attached. In some embodiments, the beads are applied to a defined area on the substrate as a slurry in a volume of less than 2 μL and then cured, for example, by phototreatment. In some embodiments, the substrate is treated with UV light at a wavelength ranging from about 145 nm to about 225 nm, thereby phototreating the polystyrene polymer chains in the substrate. In some embodiments, after curing (phototreatment), the dried sensor forms a spot on the cartridge, such as a spot having a diameter of about 2 mm. In some embodiments, a lens is positioned on the base to form a channel from the sample port to a few mm beyond the sensor (Figures 10A-10D, 11A-11D, and 12). In some embodiments, the microfluidic device (cartridge) is placed within a Steriflex W1F pouch containing a desiccant.

[0096] 10A-10D show multiple views of one embodiment of a cartridge substrate. FIG. 10A shows a top view of a cartridge base 1000. The cartridge base 1000 includes an open area 1010 containing a substrate. The cartridge substrate 1000 can further include a housing that can include ribs 1020 for handling, including for gripping the cartridge and / or inserting or removing the cartridge from a reader. The cartridge substrate 1000 can be made from any suitable material, including polystyrene. In some embodiments, the base 1000 includes a material having a dark color configured to reduce the reflected intensity of 660 nm excitation light, such as a dark gray substrate. In some embodiments, the open area 1010 of the substrate is treated with UV light at a wavelength ranging from about 145 nm to about 225 nm. In some embodiments, the light treatment renders the polystyrene polymer chains photolabile to reaction with the substrate. In some embodiments, following the light treatment, the substrate is contacted with the light-treated region of the substrate, bonding the substrate to the substrate at specific regions within the open area 1010. The substrate can include any substrate disclosed herein.

[0097] Figure 10B shows a side view of cartridge base 1000 with an open area 1010 where a substrate can be placed and includes ribs 1020. Figure 10C shows a bottom view of cartridge base 1000. Figure 10D depicts an enlarged cross-sectional view of open area 1010, depicting ribs 1030 that are crushed upon coupling of base cartridge 1000 to a lens.

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

[0099] The lens 1100 is configured with a size and shape to mate with the open area 1010 of the cartridge base 1000. Upon mating, the lens compresses the ribs 1030 of the cartridge base, thereby sealing the cartridge and thereby preventing fluid leakage from the cartridge. Additionally, mating the lens 1100 to the cartridge base 1000 forms a fluid flow path 1110 through which fluid can flow into the sample port 1105 and to the substrate.

[0100] In some embodiments, the fluid flow path is of a specific size and dimension for accurate measurement of bilirubin in a sample. In some embodiments, the surface-to-volume ratio of the fluid flow path is large enough to measure bilirubin in an undiluted sample. In some embodiments, the sample is an undiluted sample, such as an undiluted sample of whole blood, plasma, serum, urine, CSF, saliva, gastric fluid, interstitial fluid, or lymphatic fluid. In some embodiments, the cartridge is configured to receive a sample volume ranging from less than 0.5 μL to more than 10 μL, such as 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 μL.

[0101] In some embodiments, the cartridge is configured to measure the sample at equilibrium. Conventional commercially available measurement devices and / or systems can measure bilirubin that is not at equilibrium. However, the cartridge of the present invention is configured to measure bilirubin in a sample at equilibrium.

[0102] In some embodiments, the cartridge is configured to improve the accuracy of bilirubin measurement through traceability. Traceability can be performed, for example, by using a standard bilirubin sample obtained from Sigma, which is used to calibrate the probe described herein. The calibrated probe is then used to calibrate a set of bilirubin-human serum albumin complexes. The calibrated complexes are then used to calibrate cartridge lots (as described in more detail in the Examples herein).

[0103] Some embodiments provided herein relate to a custom Qiagen LR3 fluorescence reader into which a sample-containing cartridge is inserted. The reader measures fluorescence intensity at 700 nm and 819 nm, including any fluorescence from the sample itself, by scanning across the sensor using a 660 nm excitation light source and measuring fluorescence from before and after the sensor (background intensity). The reader uses the measured fluorescence intensity to form an R value and calculates the Bf concentration using equation (2). Each cartridge can be stored in a sealed pouch containing a desiccant, and a barcode containing calibration parameters is printed on the pouch and scanned by the reader. After applying a sample to the cartridge and inserting it into the reader, Bf can be displayed in approximately 90 seconds.

[0104] Using the Sensor In some embodiments, the sample used to measure unbound bilirubin is a fluid sample from a human, animal, or plant. In some embodiments, the fluid is whole blood, plasma, serum, urine, CSF, saliva, gastric juice, interstitial fluid, or lymph. In other embodiments, the determination of unbound bilirubin is performed in the cytoplasm of cells by microinjecting or otherwise transfecting the cells with a sensor, or in the extracellular medium of cells or tissue sections.

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

[0106] Unbound bilirubin sensors with zero Rm can be calibrated and used to measure [Bf] as described in U.S. Patent No. 9,529,003 and [Huber et al., Fluorescent Sensors for Quantifying Unbound Bilirubin Concentration, Clin Chem 58: 869-876, 2012]. Methods for calibrating and using probes with Rm > 0 are described for the first time herein in Equations (1-3) (calibration) and the calculation of Bf, Equation (2).

[0107] Unbound bilirubin sensors are used to measure Bf in patients at risk for bilirubin-mediated toxicity, such as 80% of all newborns, because their liver function is insufficient to eliminate excess bilirubin [Bhutani et al., Pre-discharge screening for severe neonatal hyperbilirubinemia identifies infants requiring phototherapy, J Pediatr 2013;162:47782].

[0108] The unbound bilirubin sensor is useful in patients with hemolytic diseases, patients receiving intravenous infusion of oil emulsions, patients receiving drugs that may displace bilirubin from albumin, and patients with diseases such as sepsis, which is common in premature infants, where Bf may be elevated due to a decrease in the binding affinity of bilirubin to albumin and an increase in FFA concentrations [Nogueira et al., Alterations in plasma free fatty acid concentrations in septic patients are associated with cardiac damage and reduced heart rate variability, Shock 29: 342-348, 2008], [Hegyi et al., Effect of soybean lipid infusion on unbound free fatty acids and unbound bilirubin in premature infants, J Pediatr 2017; 184: 45-50].

[0109] The unbound bilirubin sensor can be used for the measurement of Bf in patients undergoing phototherapy, blood transfusion, or other therapies designed to reduce bilirubin toxicity.

[0110] Because unbound bilirubin, rather than total bilirubin, is toxic, unbound bilirubin rather than total bilirubin may be monitored during phototherapy to ensure a significant decrease in unbound bilirubin. Total bilirubin has been shown to decrease in response to phototherapy, but the presence of bilirubin-substituting molecules, such as FFAs and certain drugs prescribed for neonates, can result in almost complete dissociation of total and unbound bilirubin. Under these conditions, virtually complete destruction of total bilirubin may be required to reduce unbound bilirubin levels to levels considered nontoxic. Thus, much lower total bilirubin levels than currently achieved with aggressive treatment may be required [Hegyi et al., "Unbound Free Fatty Acids in Preterm Infants Treated with Intralipid: Separation of Unbound Free Fatty Acids from Total Bilirubin and Potential Ineffectiveness of Phototherapy," Neonatology 2013;104:184-187 and Hegyi et al., "Effect of Soy Lipid Infusion on Unbound Free Fatty Acids and Unconjugated Bilirubin in Preterm Infants," J Pediatr 2017;184:45-50]. Furthermore, peroxidase assessment cannot be used to monitor unconjugated bilirubin during phototherapy because this test does not distinguish between photoisomers of bilirubin or conjugated bilirubin and the "native" unconjugated IX-α(Z,Z) isomer. In contrast, the unbound bilirubin measured with the UBCheck sensor described in this application is specific for the natural unconjugated IX-α(Z,Z) isomer.

[0111] The only method currently used to determine unbound bilirubin is based on horseradish peroxidase oxidation of bilirubin [Jacobsen J and Wennberg RP, Determination of unbound bilirubin in neonatal serum, Clin Chem 20:783, 1974]. Peroxidase assays are available using FDA-approved assays (Arrows Ltd, Osaka, Japan). Accurate unbound bilirubin measurements are complicated by the Arrows method [Ahlfors CE, Measurement of plasma unbound unconjugated bilirubin: Anal Biochem 279:130-135, 2000; Ahlfors et al., Sample dilution, peroxidase co-feeding, and chloride ion effects on unbound bilirubin in preterm neonates, Clin Biochem 40:261-267, 2007], which is common in jaundiced neonates. Importantly, multiple relatively large sample volumes (20–25 μL) of plasma or serum are required to measure Bf using the Arrows UB analyzer UA-2. Furthermore, the Arrows assay, which involves diluting the sample 52-fold, does not determine the equilibrium unbound bilirubin concentration, and correction for interfering substances is required using the Arrows method [Ahlfors et al., "Effects of Sample Dilution, Peroxidase Concentration, and Chloride Ion on the Measurement of Unbound Bilirubin in Premature Infants," Clinical Biochemistry 40 (2007) 261–267; Ahlfors et al., "Unbound (Free) Bilirubin: An Improved Paradigm for Assessing Neonatal Jaundice," Clinical Chemistry 55:7 1288–1299 (2009)].

[0112] Embodiments provided herein relate to a method for measuring unbound bilirubin in a single step. In some embodiments, this method overcomes the drawbacks of the peroxidase Arrows method. In some embodiments, the method uses a fluorescently labeled mutant fatty acid-binding protein (unbound bilirubin sensor), which allows for direct monitoring of equilibrium unbound bilirubin concentrations in undiluted blood samples. The probe is specific for the Z,Z isomer of unconjugated bilirubin and can bind to Z,Z isomer bilirubin with high affinity. Furthermore, the unbound bilirubin sensor can be highly specific for unbound bilirubin and does not respond to or significantly bind to free fatty acids (FFAs), bilirubin photoisomers, conjugated bilirubin, other metabolites, and most drugs present in blood. The unbound bilirubin sensor can be used to determine unbound bilirubin levels in jaundice patients, including newborns, to assess the potential risk of bilirubin neurotoxicity and thereby accurately direct treatment to prevent such toxic consequences.

[0113] In some embodiments, the method further comprises administering a treatment or therapy to a subject selected or identified as having or suffering from bilirubin neurotoxicity based on the results of the risk of bilirubin neurotoxicity. In some embodiments, the treatment or therapy is phototherapy, exchange transfusion, intravenous immunoglobulin therapy (IVIg), or drug therapy with a bilirubin inhibitor. In some embodiments, the phototherapy treatment or therapy involves exposure to a lamp emitting light in the blue-green spectrum, where exposure to light increases bilirubin excretion. In some embodiments, exchange transfusion involves repeatedly withdrawing blood and replacing it with unaffected blood, such as blood from a donor. In some embodiments, the IVIg treatment or therapy involves intravenous transfusion of blood proteins, which can reduce antibody levels in the blood of a subject suffering from bilirubin neurotoxicity. Other treatments or therapies may include treatment or inhibition of the underlying cause of bilirubin neurotoxicity, including, for example, treatment or inhibition or amelioration of bile duct obstruction, infectious causes, or genetic disorders (e.g., Crigler-Najjar and Gilbert syndromes). [Example]

[0114] The embodiments are further defined in the following examples. It should be understood that these examples are given for illustrative purposes only. From the above discussion and these examples, one skilled in the art can ascertain the features of the embodiments described herein and can make various changes and modifications to the embodiments to adapt them to various uses and conditions without departing from the spirit and scope thereof. Thus, various modifications of the embodiments, in addition to those shown and described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications also fall within the scope of the appended claims. The disclosure of each reference cited herein is incorporated herein by reference in its entirety and is for the disclosures referenced herein.

[0115] Example 1 Sequences of bilirubin-sensitive (responsive) and non-responsive probes produced by mutations in wild-type intestinal fatty acid binding protein (SEQ ID NO:1) Wild-type intestinal fatty acid binding protein (WT rIFABP) from rat has the sequence shown in SEQ ID NO:1. Table 1 shows Bf-sensitive probes with LICOR700DX-maleimide labeled with single substituted cysteines at positions 24-98 of SEQ ID NO:1. Additionally, each probe listed in Table 1 has an N-terminal MGI. The Bf-responsive probe has 14 arginine substitutions with 14 accessible lysines: 7R, 16R, 20R, 29R, 37R, 46R, 50R, 88R, 92R, 94R, 100R, 125R, 129R, and 130R (KR14) of SEQ ID NO:1. The sequence of KR14 is shown in SEQ ID NO:2. The responsive and non-responsive variants have a C-terminal double HIS tag linker, Arg Gly Ala Ala Ser His His His His His His Ser His Arg Ala Thr Pro Asn Thr Ser Pro His His His His His His His (C2XH11; SEQ ID NO: 3). Thus, each probe listed in Table 1 contains the N-terminal MGI-KR14-C2XH11, in addition to further substitutions and additions indicated in the table.

[0116] [Table 1]

[0117] [Table 2]

[0118] Example 2 Influence of non-fluorophore positions on Bf binding properties and non-mutation effects on emission spectra of non-responder proteins All responding probes labeled with LICOR700DX-maleimide and non-responding probes labeled with LICOR800CW-maleimide probes in Tables 1 and 2, respectively, have qualitatively similar dependence on Bf. The probes differ largely in protein expression, binding parameters (Kd, Ro, and Rm), and stability. The effects of different mutant proteins and fluorophore labeling positions on Kd and Rm indicate that preferred responding probes may be those with low Kd and / or low Qs (Qs = Rm / Ro), indicating the degree to which the probe is quenched by bilirubin binding (Table 3). While non-responders are not characterized by bilirubin binding parameters, the emission spectrum of 800CW-maleimide is sensitive to specific substitutions that can alter LICOR700DX-maleimide intensity. For example, in the absence of 700DX-maleimide, many initially non-responsive mutants revealed a time-dependent increase in emission at 700 nm relative to 819 nm. In some of these mutants, the 700 to 819 nm ratio increased by approximately 1% to 20% during storage at 4°C. Appropriate mutations eliminated this instability. For example, Mut:SEQ ID NO:1 B C73 in Table 2 showed the same 700 / 819 ratio (1%) on days 1 and 6.

[0119] [Table 3]

[0120] Example 3 Changes in fluorescence of bilirubin-responsive (700 nm) and non-responsive (800 nm) probes and their ratio (700 / 800) Figure 1 shows the intensity and intensity ratio of Bf sensors composed of the bilirubin-responsive LICOR700DX maleimide Mut:SEQ ID NO:2-76C probe (Table 1) and the bilirubin-unresponsive LICOR800CW maleimide Mut:SEQ ID NO:1 B-73C probe (Table 2), both of which are free in aqueous buffer and exhibit increasing BT (total bilirubin), which at the concentrations used is primarily unbound bilirubin (Bf). Measurements of 710 and 805 nm intensity at 675 nm excitation were performed in a Horiba Fluorolog 2 at probe concentrations of 1.5 nM and 10 nM, respectively. The results show a monotonic decrease in LICOR700DX maleimide intensity (measured at 710 nm) attached to bilirubin-sensitive iLBPs and a lack of intensity change (cv = 2.5%) for LICOR800CW maleimide (measured at 805 nm) attached to unresponsive iLBPs. The inserts exhibit a behavior in the 710 / 805 ratio.

[0121] Example 4 Calibration of free NIR ratio sensors by cuvette fluorescence method A Bf sensor consisting of LICOR700DX maleimide-labeled bilirubin-sensitive iLBP (Mut: SEQ ID NO: 2-76C) and LICOR800CW maleimide-unresponsive iLBP (Mut: SEQ ID NO: 1B-73C) from Tables 1 and 2, respectively, free in aqueous buffer, was titrated with increasing concentrations of Sigma bilirubin (Cat: B4126) solubilized in aqueous buffer at pH 12 (Figure 2). BT concentrations were determined by absorbance at 441 nm. At each bilirubin concentration, measurements of fluorescence excited at 660 nm and emission at 700 and 805 nm were used to determine the ratio (R) of background minus 700-805 emission. The total bilirubin BT in the cuvette at each step of the titration is the total free bilirubin, excluding the portion that is soluble and binds to the sensor. A least-squares fit to the obtained titration curve (R vs. BT) (Figure 2) is performed using equation (1), which considers the probe-bound bilirubin. This procedure calibrates the free probe by determining its calibration parameters (Kd, Rm, and Ro). The results of the fit in this case were a Kd of 12.4 ± 0.4 nM, an Rm of 0.42 with an Ro of 3.02, and a Qs of 0.139 ± 0.007 Rm / Ro.

[0122] Example 5 Bilirubin-albumin (HSA) complex calibration To calibrate the Bf test cartridge, a series of highly buffered aqueous samples with increasing Bf concentrations are prepared. Highly buffered Bf samples are those whose Bf concentration is not affected by bilirubin binding to the cartridge channel walls or by binding to the dried bilirubin probe in the channel. A complex of bilirubin bound to HSA (BR:HSA) at a concentration of 400-1000 μM is sufficient to buffer or clamp the Bf concentration in the test cartridge, at which Bf remains unchanged from the calibrated level when bilirubin wall binding is negligible. BR:HSA complexes are typically prepared at a bilirubin-to-HSA molar ratio of 0.1-1.0 in steps of 0.05-0.1. The complexes are calibrated by measuring the Bf concentration produced by each complex in a cuvette by adding approximately 1-20 nM of a free Bf sensor, calibrated as in Example 4, to each undiluted complex. Typically, Bf values ​​increase exponentially with increasing BR:HSA from 0 to 0.9, where, starting at 0.1, Bf increases from 2 to 300 nM (Figure 3).

[0123] Example 6 Calibration of test cartridges with BR:HSA complex A significant number of test cartridges, called "lots," are manufactured in a given period, e.g., 500-2000 or more, and are calibrated by randomly selecting a sufficient number of cartridges so that each bilirubin-albumin conjugate can be applied to replicate cartridges. A single calibration of a lot requires at least three measurements of each conjugate, which ranges from 33 to 60 cartridges, depending on the BR:HSA step size. Each cartridge is used for only a single measurement and then discarded. The measured R values ​​from each cartridge and each conjugate are fitted by least-squares fitting using Equation 2 to obtain the Ro, Kd, ​​and Rm of the cartridge lot. An example of cartridge calibration using the BR:HSA conjugate reveals that the apparent Kd and Qs can be similar to or even greater than the free probe parameters (Figure 3). The increase in Kd may be the result of probe immobilization and interaction with adjacent surfaces during drying of the probe-beads on the cartridge.

[0124] Example 7 Single-Step Disposable Cartridge The single-step disposable cartridge is an essential component of this invention. The cartridge contains all the components necessary for measuring Bf upon addition of a patient blood sample and insertion of the cartridge into the reader. Because newborns weigh as little as 400 g, it is essential to use very small blood volumes for Bf measurement. The minimum sample volume for the disposable cartridge is 3.8 μl. Furthermore, because whole blood, as well as plasma, serum, and other fluids, are measured, the optical path lengths of the excitation and emission optical paths must be as short as possible. These considerations led to the development of the cartridge shown in Figures 10A-10D and 11A-11D. In this device, the sample is added to a port and fills a groove formed by an acrylic lens snapped onto the cartridge base, which contains a bilirubin sensor spot in the center of the groove. The resulting groove is 0.1 mm high, 2.7 mm wide, and 14 mm long, accommodating a 3.8 μL sample volume.

[0125] Maintaining these dimensions requires high tolerances in molding. A key and novel component of this device is the ability to form a sealed groove on the dried Bf bilirubin sensor-polystyrene beads by simply snapping a clear acrylic lens onto the polystyrene base (Figures 10A-10D, 11A-11D, and Figure 12). This is achieved by creating an oval groove in the base, which, once snapped into place, presses against a crushed rib beneath the surface of the base, forming a sealed channel. To achieve rapid capillary flow of the sample over the bilirubin sensor spot, the acrylic lens is treated with O2 plasma at a level that increases the hydrophilicity of the lens without compromising its optical transparency in the NIR. Additionally, the polystyrene base is treated with UV radiation to increase its hydrophilicity, enhance rapid capillary flow, and adjust the sensor spot size. UV irradiation is also important for allowing the bilirubin sensor polystyrene beads to firmly adhere to the polystyrene substrate. UV irradiation at 254 nm has been shown to disrupt polymer bonds, thereby allowing intercalation of polymer chains from opposing surfaces and increasing the binding affinity between surfaces. [Maeda et al., Adhesion and Friction Mechanisms of Polymers on Polymer Surfaces, Science (2002) 297, 379-382]. Irradiation from a 254 nm source increases the binding affinity between polystyrene surfaces. However, the degree of adhesion is highly sensitive to the lifetime of the 254 nm bulb, and irradiation times can be longer than 60 minutes. The present invention is the discovery that typical 254 nm bulbs also produce low levels of 185 nm radiation, suggesting that 185 nm radiation is the primary cause of increased polymer adhesion. Switching to an 185 nm light source shortens exposure times to a few seconds or minutes, depending on the irradiation distance. Furthermore, spotting the sensor onto an 185 ± 40 nm irradiation cartridge results in a more uniform spot shape.Thus, in some embodiments, the wavelength is in the range of about 145 nm to about 225 nm, such as 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, or 225 nm, or a wavelength within a range defined by any two of the foregoing values.

[0126] The Bf sensor attached to the cartridge surface is incompletely quenched by bilirubin, in contrast to the fully quenched sensor described in U.S. Pat. No. 9,529,003. For example, U.S. Pat. No. 9,529,003 describes a bilirubin probe with a HIS tag and a polystyrene tag (PS) attached to Ni-agarose beads immobilized in the wells of a "disposable cartridge," but does not provide details of such a cartridge. As shown in Figure 8 of U.S. Pat. No. 9,529,003, the Qs (Rm / Ro) of the probe was 0.02 ± 0.03 (Rm = 0), indicating a fully quenched Bf sensor. The finite-limiting quenching (Rm > 0) of the present disclosure uses a different analysis to determine the exact Bf level. The analysis described herein is mathematically detailed in Equations 1 and 2, which reduce to Equations 2 and 3 of U.S. Pat. No. 9,529,003 for Rm = 0.

[0127] U.S. Patent No. 9,529,003 describes measuring Bf in whole blood in a microfluidic device in which a magnet is used to concentrate Bf sensors on iron-containing polystyrene beads at the bottom of a multiwell plate. This configuration is impractical for single-step assays, in which the sample cartridge is sealed within a desiccant-containing pouch, and the sensor must therefore be tightly bound to the cartridge surface. Instead, in this disclosure, a slurry containing bilirubin-sensitive and -unresponsive iLBP mutants attached to polystyrene beads is prepared, each with a different NIR fluorophore labeled at a cysteine ​​residue, all in an aqueous buffer solution. Small droplets (<2 μl) of this slurry are dispensed onto a UV-treated cartridge, dried, and placed in a desiccant-containing pouch.

[0128] Example 8 Determination of equilibrium Bf concentration Bf in blood samples is in equilibrium due to the binding and dissociation of bilirubin with albumin. Typical albumin concentrations are 400-600 μM, and the equilibrium dissociation constant (Kd) of bilirubin for adult human albumin is approximately 20 nM for the high affinity site. A bilirubin-albumin molar ratio of 0.5 at equilibrium produces approximately 20 nM of Bf. The estimated capacity of the Bf sensor in each cartridge is 2 x 10 -13 Therefore, in a 5 μl sample, the sensor concentration is 40 nM. This means that at best, the sensor will react with less than 40 nM bilirubin, or about 1.6 x 10 of 250 μM total bilirubin bound to 500 μM albumin. -4This means that the bilirubin binds to the albumin. Because Bf is approximately Kd*BT / albumin, small changes in BT have a negligible effect on Bf. Because the sensor's effect on the actual albumin-buffered Bf equilibrium concentration is negligible, UBCheck approximates the equilibrium Bf concentration. It is important not to dilute the sample due to bilirubin-albumin complexes, but to produce a highly buffered Bf level that remains undisturbed by the amount bound by the sensor. Other methods for measuring Bf in blood samples do not measure at equilibrium. Most importantly, the peroxidase method, which has been in use for 45 years and is implemented by the FDA-certified Arrows UB analyzer, does not yield Bf at equilibrium [Jacobsen et al., Determination of Unbound Bilirubin in Neonatal Plasma, Clin Chem (1974) 20, 183]. This is due in part to the large sample dilution (42-52-fold) in the FDA-certified Arrows method and the peroxidase method, which oxidizes a substantial fraction of Bf. By measuring samples at different peroxidase concentrations and extrapolating to zero peroxidase concentration, better estimates of Bf closer to equilibrium can be obtained [Ahlfors et al., Unbound (Free) Bilirubin: An Improved Paradigm for Assessing Neonatal Jaundice, Clin Chem (2009) 55:7 1288-1299]. However, this method is not FDA-approved and ultimately only provides a better equilibrium approximation. However, a more recent method for measuring Bf has been proposed that does not measure equilibrium and obtains Bf concentrations several orders of magnitude greater than those obtained with the present invention or the peroxidase method [Bell et al., Paper-Based Potentiometric Detection of Free Bilirubin in Serum, Biosensors and Bioelectronics, 126 (2019) 115-121]. This method destroys Bf in equilibrium with albumin by using a filter and electromotive force to separate bilirubin from albumin.

[0129] Example 9 Effect of dilution on equilibrium - When bilirubin-albumin substitution increases HSA Kd UBCheck most closely measures equilibrium Bf, thereby most closely approximating steady-state Bf levels in the circulation. A direct illustration of the buffering capacity of the bilirubin:albumin complex is the response of the sensor in the cartridge with and without albumin. Addition of free bilirubin at a concentration of 100 nM to the cartridge results in a Bf of ≤1 nM as detected by the sensor, while addition of 1000 nM free bilirubin detects a Bf of 50 nM. This loss of free bilirubin is due to its binding to the polystyrene and acrylic surfaces that define the sample groove in the cartridge, particularly due to the large surface-to-volume ratio of the sample groove (20).

[0130] Even without surface binding, equilibrium Bf concentration depends on sample dilution due to the kinetics of the bilirubin-HSA reaction. Furthermore, the effect of dilution is amplified in the presence of bilirubin displacers from albumin, which effectively reduce the binding affinity of HSA for bilirubin. This is illustrated by the measurement of Bf as a function of bilirubin-HSA complexes at molar ratios of 0.1 to 0.5 when oleic acid is added to HSA at a molar ratio of 6 oleic acid to 1 HSA (Figure 4). Starting at an HSA concentration of 550 μM, Bf concentration decreases by more than an order of magnitude with a 42-fold dilution (the dilution used by the original Arrows UB analyzer). Oleic acid is a potent FFA displacer and is a key 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 the Bf-responsive and non-responsive probes to Ni-polystyrene beads, the peptides listed in Table 4 were added to the beads and attached to the Ni-NTA beads via the dual HIS tag. The combined probe and peptide beads were then spotted onto a cartridge. The effect of whole blood hemolysate was tested by titrating bilirubin-spiked neonatal serum with increasing hemoglobin concentrations. Bf concentrations were measured before and after hemoglobin titration, revealing that the Bf concentration relative to zero hemoglobin was unaffected by hemoglobin concentrations as high as 4 g / L (Figure 5).

[0132] [Table 4]

[0133] Example 11 Effects of medications prescribed for newborns The embodiments described herein relate to measuring Bf concentrations in newborns. Because infants in NICUs frequently receive medication, premature infants, who are at high risk for bilirubin neurotoxicity, are of particular concern. [Hsieh et al., Drug Use in the Neonatal Intensive Care Unit, Am J Perinatol (2014) 31, 811-822]. Table 5 shows that certain drugs, among those most frequently prescribed in NICUs, are potent displacers of bilirubin from albumin and interference, as determined by measuring Bf using the one-step cartridge of Example 7. The drug rank indicates frequency of use in NICUs, starting with ampicillin, which is ranked 1 as the most prescribed drug. Elements in the "Displacement or Interference" column are blank if the drug has no effect. Displacers are positive, indicating a three-fold increase in drug concentration compared to no drug. A negative value for spironolactone indicates interference with the Bf sensor. As shown in Table 5, several drugs are strong displacers, with spironolactone being the only NICU drug found to interfere with Bf assessment. Figure 6 shows the effect on Bf as a function of prescribed drug concentration (low, medium, high, and 3x high) for drugs in Table 5 that are strong displacers of bilirubin from albumin.

[0134] [Table 5]

[0135] The increase in Bf caused by these drugs can significantly exceed the upper limit of normal for NICU infants, potentially resulting in serious health consequences. Furthermore, the increase in Bf caused by these drugs, as described in Example 7, can significantly exceed the upper limit of normal for NICU infants, potentially resulting in serious health consequences. Furthermore, similar to the effect of FFA (oleic acid) as described in Example 7, the effect of dilution significantly influences the degree of drug-induced displacement. Table 6 demonstrates this effect by comparing Bf levels in adult serum spiked with bilirubin and then 1.12 and 3.76 mM cefazolin. Bf measurements were performed using the Arrows UB2 analyzer (52-fold dilution) and UBCheck (undiluted). As Table 6 shows, Bf in the presence of 1.12 and 3.76 mM cefazolin was approximately 3- and 5-fold greater in UBCheck than in Arrows.

[0136] [Table 6]

[0137] Example 12 Reduced interference from conjugated bilirubin Potential interference between conjugated bilirubin and the bilirubin probe was determined by measuring the effect of ditaurobilirubin on the ability of UBCheck to accurately measure Bf. UBCheck results were also compared to measurements using the Arrows UB analyzer UA-2 method. Measurements are performed by spiking newborn serum with unconjugated bilirubin to obtain a Bf of approximately 10 nM, then titrating the spiked sample to 20 mg / dL with ditaurobilirubin (Figure 7). At each step, Bf is measured by UBCheck and Arrows, and direct (bound) bilirubin concentration is determined using the Sigma direct bilirubin kit. With zero ditaurobilirubin, both UBCheck and Arrows yield a Bf of 10 nM, while direct bilirubin measurements yield a non-zero value of approximately 0.5 mg / dL (Figure 7). As ditaurobilirubin concentration increases, UBCheck remains unchanged, whereas Arrows increases starting at approximately 0.5 mg / dL ditaurobilirubin and then rapidly increases up to 4 mg / dL ditaurobilirubin, where Arrows estimates saturate at Bf ≥ 50 nM. In contrast, UBCheck only begins to increase at 4 mg / dL ditaurobilirubin, where its Bf increases 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. Thus, UBCheck has better specificity for Bf than Arrows, even when the Arrows sample is diluted 52-fold, whereas the UBCheck sample is undiluted.

[0138] Example 13 Lack of interference from bilirubin photoisomers Neonatal hyperbilirubinemia is most frequently treated with blue-green light therapy, which photoisomerizes albumin-bound bilirubin. [Newman et al., Neonatal Hyperbilirubinemia and Long-Term Outcome: Another Observation from the Collaborative Perinatal Project, Pediatrics (1993) 92, 651-657; Ennery JF, Blue Light, Green Light, White Light, Other Lights: Treatment of Neonatal Jaundice, Clinical Practice in Perinatal Medicine (1990) 17, 467-481]. The bilirubin photoisomers (4Z,15E), (4E,15Z), and lumirubin are much more soluble and therefore more readily excreted than the native bilirubin IXa (4Z,15Z) molecule. Phototherapy readily reduces the Z,Z isomer, thereby effectively treating neonatal hyperbilirubinemia. Photoisomers can also be generated by exposure to ambient light [McDonagh et al., Photoisomers: Confounding Factors in Clinical Peroxidase Measurement of Unbound Bilirubin? Pediatrics (2009) 123, 67-76]. As shown in Figures 9A and 9B, the Bf assay detects only the Z,Z isomer of bilirubin, while the Arrows assay is sensitive to Z,Z and all photoisomers. However, only the Z,Z isomer, not the photoisomer, is toxic [Jasparova et al., Photoisomers of Bilirubin (2016) PLoS ONE 11(2):e0148126. doi: 10.1371 / journal.pone.0148126]. Serum samples from healthy newborns who did not undergo phototherapy but whose HPLC analysis indicates substantial levels of photoisomers (presumably due to serum exposure to ambient light) also show more than double the Bf levels by Arrows compared to the UBCheck Bf assessment, as shown in Figure 9A. As shown in Figure 9B, adult serum samples were spiked with bilirubin and then exposed to a phototherapy lamp (Natus Neoblue) for 5 hours. The top panel shows the results of the relative intensities of the Z,Z, and 3 photoisomers determined from multiple HPLC scans over the 5-hour exposure. This shows that Z,Z monotonically decreases to low levels, while the photoisomers peak at approximately 1.5–2 hours and then decrease toward zero. The bottom panel shows the results of two samples spiked with bilirubin, whose Bf levels were 8 and 12 nM before phototherapy by both Arrows and Bf assessment.In contrast to Arrows, the Bf evaluation followed a monotonic decrease of Z,Z toward zero, whereas Arrows Bf increased rapidly by 1 h, reaching its saturation level of 50 nM. Thus, Arrows is highly sensitive to photoisomers.

[0139] Bleaching serum with a phototherapy lamp, as shown in Figure 9B, reveals that the Bf concentration measured by UBCheck is essentially zero after 5 hours. Such experiments allow for quantification at the limit of detection and further demonstrate that in serum or plasma lacking bilirubin, UBCheck is not affected by any other blood-containing molecules, including other hydrophobic metabolites such as fatty acids, other lipids, peptides, nucleic acids, etc. Importantly, given that phototherapy treatments for infants are typically used for much longer periods (typically 24-72 hours), the light from the phototherapy lamp is unlikely to be destructive to other blood components. UBCheck's zero response tendency indicates that the assessment is not sensitive to all natural blood metabolites, including FFAs, peptides, nucleic acids, or any other natural blood components, so UBCheck is not only insensitive to photoisomers.

[0140] Example 14 Lack of interference from intralipids and triglycerides Figures 8A-8B show that Intralipid, as triglyceride, can decrease Bf (Figure 8) and, upon lipolysis, generates unbound FFAs that can increase Bf by displacing bilirubin from albumin (Figure 8B). In Figure 8A, a neonatal serum sample spiked with bilirubin to produce a Bf of 24 nM was titrated with triglyceride (Intralipid) to 10 mM, which resulted in a monotonic decrease in Bf to 18 nM. These results appear to correspond to Intralipid infusion without heparin. The decrease in Bf was accurately monitored by the UBCheck method, indicating that triglycerides create a sink for bilirubin and are therefore not an interfering substance. Figure 8B shows the results of approximately 100 premature infants who received Intralipid infusions in the presence of heparin, starting at 1 g / kg / day (IL1) and increasing to 2 g / kg / day (IL2) and 3 g / kg / day (IL3). With each Intralipid step, Bf levels increased due to heparin-activated lipase production of unbound FFAs (FFAu), which displaced bilirubin from albumin. These results are consistent with the absence of triglyceride or FFA interference with Bf measurement. In other words, turbidity (light scattering) did not affect the assessment due to at least the following reasons: 1) the fluorescence excitation (660 nm) and emission (700 and 819 nm) are in the near-infrared (light scattering decreases with increasing wavelength), 2) the cartridge path length is 0.1 mm, and 3) if the 700 / 819 ratio were affected by scattering, the 700 intensity would decrease more than the 819 intensity, thereby decreasing the R value and thereby increasing Bf rather than decreasing Bf as in Figure 8A. In contrast, the peroxidase method performed by Arrows (the measurement is performed at 460 nm) is highly affected by scattering, as Arrows reports an increase in total bilirubin with increasing intralipid levels.

[0141] Example 15 Lack of interference from FFAu Lipid infusions, such as interlalipid in the presence of heparin, can produce exceptionally high concentrations of unbound free fatty acids (FFAu), much of which displaces bilirubin from albumin (Figure 4). In neonates receiving increasing concentrations of interlalipid, FFAu levels can increase beyond 100 nM [Hegyi T. et al., Effects of soybean lipid infusion on unbound free fatty acids and unbound bilirubin in premature infants, (2017) J Pediatr 184, 45-50e41]. However, the results in Figure 4 reveal that when the molar ratio of oleic acid to albumin is 6:1, the unbound oleic acid concentration is greater than 500 nM [Richieri et al., Interaction of long-chain fatty acids with albumin: measurement of free fatty acid levels with the fluorescent probe ADIFAB, Biochemistry 32 7574-7580 (1993)], but by increasing Bf, only the increase from bilirubin to albumin affects the sensor, so FFAs do not interfere with the Bf sensor.

[0142] Example 16 Analytical specifications and determination of Bf in bilirubin-spiked and non-spiked human serum / plasma Analytical specifications for the UBCheck evaluation were determined using multiple cartridge lots and two or more readers according to CLSI guidelines. Results include the limit of blank (LOD = 0.7 nM), limit of detection (LOD = 0.9 nM), and limit of quantitation (LOQ = 0.9 nM), determined by complete bleaching of serum samples. A three-center precision study was performed using three readers, three different cartridge lots, and bilirubin-spiked neonatal and adult serum samples. The average results across the three readers and cartridge lots were as follows (UB level (nM) and CV (%)): 4.4, 9%; 8.0, 7%; 11.4, 8%; 19.1, 7%; and 38.2, 8%.

[0143] Pooled human plasma (Golden West Biologicals) containing 620 μM albumin was spiked with bilirubin to produce plasma samples with bilirubin / albumin molar ratios of approximately 0 to 0.9. Measurements of [Bf] were also performed on the same samples using a peroxidase method implemented on an Arrows Bf analyzer. These results demonstrate that the bilirubin probe response is entirely due to its interaction with bilirubin and that the bilirubin probe does not respond to other metabolites present in human blood samples. Furthermore, agreement with the peroxidase method and predictions of the bilirubin-albumin equilibrium also demonstrates that the probe produces accurate unbound bilirubin concentrations.

[0144] As used herein, section headings are for organizational purposes only and should not be construed as limiting the subject matter described in any way. All literature and similar materials cited in this application, including but not limited to patents, patent applications, papers, books, articles, and internet web pages, are expressly incorporated by reference in their entirety for any purpose, including the disclosures specifically referenced herein. If the definitions of terms in incorporated references appear to differ from the definitions provided in this disclosure, the definitions provided in this disclosure shall control. It is understood that before the temperatures, concentrations, times, etc. discussed in this disclosure, there is an implied "about" so that minor deviations and minor deviations are within the scope of the present disclosure herein.

[0145] In this application, the use of the singular includes the plural unless specifically stated otherwise, and the words "comprise," "comprises," "comprising," "contains," "contains," "containing," and "include" are not intended to be limiting.

[0146] As used in this specification and claims, the singular forms "a," "an," and "the" include 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 appreciate that the present disclosure extends beyond the specifically disclosed embodiments to other alternative embodiments and / or uses thereof, as well as obvious modifications and equivalents thereof. In addition, while several variations have been shown and described in detail, other modifications that are within the scope of the present disclosure will be readily apparent to those skilled in the art based on this disclosure. It is also contemplated that various combinations or subcombinations of specific features and aspects of the embodiments can be made and still fall within the scope of the present disclosure. It should be understood that various features and aspects of the disclosed embodiments can be combined with or substituted for one another to form various modes or embodiments. Accordingly, it is not intended that the scope of the present disclosure as described herein should be limited by the specific disclosed embodiments described above.

[0148] It should be understood, however, that this detailed description, while indicating various embodiments, is given by way of example only, since various changes and modifications within the spirit and scope thereof will become apparent to those skilled in the art.

[0149] The terms used in the descriptions presented herein are not intended to be limiting or construed in a limiting manner. Rather, the terms are merely utilized in conjunction with detailed descriptions of embodiments of systems, methods, and related components. Furthermore, embodiments may include several novel features, no single one of which is solely responsible for its desirable attributes or considered essential to practicing the embodiments described herein.

Claims

1. A sensor for measuring free bilirubin in a sample, the sensor comprising: a bilirubin-responsive probe labeled with a first fluorophore, the bilirubin-responsive probe comprising a first intracellular lipid-binding protein (iLBP), wherein the first iLBP has a peptide sequence comprising SEQ ID NO: 1; Arg replacing the 14th accessible lysine (KR14 as set forth in SEQ ID NO:2); a C-terminal double His tag linker (C2XH11) having the sequence set forth in SEQ ID NO: 3; N-terminally added MGI; and Up to 62 amino acid substitutions and additions, including a single cysteine; Including, wherein the first fluorophore is LICOR700DX maleimide or LICOR800CW maleimide attached to a single cysteine ​​substitution; the sensor comprises a non-responsive probe labeled with a second fluorophore, the non-responsive probe comprising a second iLBP, wherein the second iLBP has a peptide sequence comprising SEQ ID NO: 1; substitutions at positions 72, 73, 74, 126, and 131; a substitution at any one of positions 27, 31, 33, 54, 73, 74, 76, or 98 to Cys; no more than three additional amino acid substitutions; and a C-terminal double His tag linker (C2XH11) having the sequence set forth in SEQ ID NO: 3; Including, wherein the first and second fluorophores are excited at the same wavelength and the first and second fluorophores fluoresce at different wavelengths.

2. 2. The sensor of claim 1, wherein the bilirubin-responsive probe comprises the sequence of any one of the probes set forth in Table 1 (SEQ ID NOs: 4-15).

3. 3. The sensor of claim 1, wherein the non-responsive probe comprises the sequence of any one of the probes listed in Table 2 (SEQ ID NO: 16-28).

4. The sensor according to any one of claims 1 to 3, wherein the first fluorophore and the second fluorophore are different fluorophores.

5. The sensor of any one of claims 1 to 4, wherein the bilirubin-responsive probe comprises a single cysteine ​​to which the first fluorophore is attached.

6. The sensor of any one of claims 1 to 5, wherein the non-responsive probe comprises a single cysteine ​​to which the second fluorophore is attached.

7. The sensor of any one of claims 1 to 6, wherein the bilirubin-responsive probe is configured to bind to the unconjugated IX-α(Z,Z) isomer of bilirubin.

8. The sensor of any one of claims 1 to 7, wherein the bilirubin-responsive probe is configured to minimally bind conjugated bilirubin (less than 4 mg / dl).

9. 9. The sensor of claim 1, wherein the bilirubin-responsive probe is configured to not bind to Z,E or E,Z photoisomers of bilirubin, lumirubin, fatty acids, any other naturally occurring blood components, and / or neonatal drugs.

10. 10. The sensor of claim 9, wherein the neonatal drug is not spironolactone.

11. The sensor of any one of claims 1 to 10, wherein the non-responsive probe does not bind to the unconjugated IX-α(Z,Z) isomer of bilirubin or to conjugated bilirubin.

12. 12. The sensor of claim 1, wherein the non-responsive probe is configured to not bind to the Z,E or E,Z photoisomers of bilirubin, lumirubin, fatty acids, any other naturally occurring blood components, and / or neonatal drugs.

13. 13. The sensor of any one of claims 1 to 12, wherein when the first fluorophore is LICOR700DX maleimide, the second fluorophore is LICOR800CW maleimide attached to a cysteine ​​substitution, and when the first fluorophore is LICOR800CW maleimide, the second fluorophore is LICOR700DX maleimide.

14. The sensor of any one of claims 1 to 13, wherein the single cysteine ​​substitution is at position 22, 24, 25, 26, 27, 29, 30, 33, 54, 74, 76, 97, or 98 of SEQ ID NO:

1.

15. 15. The sensor of claim 1, wherein the emission intensity of the first fluorophore or the second fluorophore is not affected by the absorbance of a blood component selected from bilirubin and hemoglobin.

16. The sensor of any one of claims 1 to 15, wherein the bilirubin-responsive probe or the non-responsive probe further comprises at least one linker.

17. A composition comprising the sensor according to any one of claims 1 to 16.

18. 1. A composition comprising a free bilirubin (Bf) sensor, the Bf sensor comprising: a first intracellular lipid-binding protein (iLBP) that binds bilirubin and is labeled with a first fluorophore, wherein the first fluorophore is LICOR700DX maleimide or LICOR800CW maleimide attached to a single cysteine ​​substitution; a second iLBP that does not bind to bilirubin and is labeled with a second fluorophore, wherein the second fluorophore is not bound to the first iLBP; Including, wherein the first iLBP has a peptide sequence comprising SEQ ID NO: 1; Arg replacing the 14th accessible lysine (KR14 as set forth in SEQ ID NO:2); a C-terminal double His tag linker (C2XH11) having the sequence set forth in SEQ ID NO: 3; N-terminally added MGI; and Up to 62 amino acid substitutions and additions, including a single cysteine; Including, wherein the second iLBP has a peptide sequence comprising SEQ ID NO: 1; substitutions at positions 72, 73, 74, 126, and 131; a substitution at any one of positions 27, 31, 33, 54, 73, 74, 76, or 98 to Cys; no more than three additional amino acid substitutions; and a C-terminal double His tag linker (C2XH11) having the sequence set forth in SEQ ID NO: 3; Including, wherein the first fluorophore and the second fluorophore are excited at the same wavelength, the emission wavelengths of the first fluorophore and the second fluorophore are different, and the second fluorophore does not change its emission in the presence of bilirubin.

19. 19. The composition of claim 18, wherein the first fluorophore is LICOR700DX maleimide and the second fluorophore is LICOR800CW maleimide, or the first fluorophore is LICOR800CW maleimide and the second fluorophore is LICOR700DX maleimide.

20. 20. The composition of claim 18 or 19, wherein the change in the ratio of fluorescence indices is measured at two different wavelengths and used to determine the concentration of unbound bilirubin.

21. 21. The composition of any one of claims 18 to 20, wherein the emission intensity of the first fluorophore or the second fluorophore is not affected by the absorbance of a blood component selected from bilirubin and hemoglobin.

22. A solid substrate comprising the sensor of any one of claims 1 to 16 or the composition of claim 17, wherein the bilirubin-responsive probe and / or the non-responsive probe is attached to a solid substrate.

23. 23. The solid substrate of claim 22, wherein the solid substrate is Ni-polystyrene, Ni-latex, or Ni-agarose beads.

24. 24. The solid substrate of claim 23, wherein the Ni-polystyrene, Ni-latex, or Ni-agarose beads contain iron.

25. 25. The solid substrate of any one of claims 22 to 24, wherein the bilirubin-responsive probe comprises the 7R 16R20R 29R 37R46R 50R 88R92R 94R 100R125R 129R and 130R (KR14) substitutions set forth in SEQ ID NO:

2.

26. The solid substrate of any one of claims 22 to 25, wherein the bilirubin-responsive probe and / or the non-responsive probe comprises a tag, and the solid substrate comprises a receptor for the tag.

27. 27. The solid substrate of claim 26, wherein the tag comprises one or more of a His-tag, biotin, Flag-epitope, c-myc epitope, HA-tag, glutathione-S-transferase (GST), maltose binding protein (MBP), chitin binding domain (CBD), thioredoxin, β-galactosidase, VSV glycoprotein, calmodulin binding protein, polystyrene (PS) hydrophobic tag, or metal affinity tag.

28. 28. The solid substrate of claim 26 or 27, wherein the tag is a polyhistidine tag and the solid substrate comprises an immobilized metal chelate.

29. The solid substrate of any one of claims 22 to 28, wherein the first fluorophore is attached to a cysteine ​​residue on the bilirubin-responsive probe.

30. 30. The solid substrate of any one of claims 22 to 29, wherein the second fluorophore is attached to a cysteine ​​residue on the non-responsive probe.

31. 1. A method of calibrating a bilirubin sensor to measure Kd and Rm, comprising: mixing the sensor of any one of claims 1 to 16 with an aqueous sample of known concentration of bilirubin (BT); 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 sample background-subtracted fluorescence intensity from the first fluorophore at wavelength λ, and I λ２ is the fluorescence intensity from the second fluorophore at wavelength λ2 with background subtracted from the sample, Ro is the ratio in the absence of bilirubin, BT is the total bilirubin concentration, PT is the probe concentration, and r is the I of the bilirubin probe fluorophore in the absence of the second fluorophore. 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:

32. 1. A method for measuring the concentration of free bilirubin [Bf] in a sample, comprising: applying a sample to the sensor according to any one of claims 1 to 16; measuring the fluorescence of the sample; and determining the concentration of [Bf] from the measured fluorescence; A method comprising:

33. The following equation (1) is used to calibrate the sensor, and the following equation (2) is used to determine [Bf]: [Equation 2] [Equation 3] where R is the measured fluorescence ratio ((I λ１ / I λ2 ), where Iλ is the fluorescence intensity from the first fluorophore at wavelength λ, Iλ is the fluorescence intensity from the second fluorophore at wavelength λ, 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 33. The method of claim 32, wherein Kd is the dissociation constant, Rm is the minimum R value at ∞Bf, and Rm is the R at bilirubin saturation of the probe.

34. 34. The method of claim 32 or 33, wherein the sample is mixed with one or more carrier macromolecules for the bilirubin.

35. 35. The method of claim 34, wherein the one or more carrier macromolecules comprise albumin, a lipid-binding protein, a lipid vesicle, or a cyclodextrin.

36. The method of any one of claims 32 to 35, wherein the sensor is attached to a solid support.

37. 37. The method of any one of claims 32 to 36, wherein the Bf concentration is determined using a disposable microfluidic device.

38. The method of any one of claims 32 to 37, wherein the sample is of human, animal, or plant origin.

39. The method of any one of claims 32 to 38, wherein the sample is whole blood, plasma, serum, urine, CSF, saliva, gastric juice, interstitial fluid, or lymph.

40. 40. The method of any one of claims 32 to 39, wherein the sample is from a patient receiving an intravenous infusion of an oil emulsion.

41. 41. The method of any one of claims 32 to 40, wherein the sample is from a patient receiving a drug that displaces bilirubin from albumin and / or such patient may produce molecules that displace bilirubin from albumin from an infused oil emulsion, from disease or stress.

42. 42. The method of any one of claims 32 to 41, wherein the sample is from a patient undergoing phototherapy, blood transfusion or other therapy that reduces bilirubin levels.

43. 43. The method of any one of claims 32 to 42, wherein Ro is obtained by photobleaching the sample, thereby obtaining a zero level measurement.

44. 1. A cartridge for measuring free bilirubin in a sample, comprising: substrate; a lens configured to couple to the substrate and including a sample port for receiving a sample; and a substrate having a bilirubin-responsive probe, a non-responsive probe, and an anti-hemoglobin peptide, wherein the bilirubin-responsive probe, the non-responsive probe, and the anti-hemoglobin peptide are immobilized on the substrate; Including, the bilirubin-responsive probe is labeled with a first fluorophore, the bilirubin-responsive probe comprises a first intracellular lipid-binding protein (iLBP), wherein the first iLBP has a peptide sequence comprising SEQ ID NO: 1; Arg replacing the 14th accessible lysine (KR14 as set forth in SEQ ID NO:2); a C-terminal double His tag linker (C2XH11) having the sequence set forth in SEQ ID NO: 3; N-terminally added MGI; and Up to 62 amino acid substitutions and additions, including a single cysteine; wherein said first fluorophore is LICOR700DX maleimide or LICOR800CW maleimide attached to a single cysteine ​​substitution; the non-responsive probe is labeled with a second fluorophore, and the non-responsive probe comprises a second iLBP, wherein the second iLBP has a peptide sequence comprising SEQ ID NO: 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）； Including, wherein said first and second fluorophores are excited at the same wavelength and said first and second fluorophores fluoresce at different wavelengths; A cartridge in which the substrate is treated with UV light at a wavelength in the range of 145 nm to 225 nm, whereby the polystyrene polymer chains of the phototreated substrate are linked to the polymer chains of the base material.

45. 45. The cartridge of claim 44, wherein the substrate is a polystyrene substrate.

46. 46. ​​A cartridge according to claim 44 or 45, wherein the substrate comprises a material having a dark colour configured to reduce the reflected intensity of 660 nm excitation light.

47. The cartridge according to any one of claims 44 to 46, wherein the lens is an acrylic lens.

48. The lens is O ２ A cartridge according to any one of claims 44 to 47 which is treated with plasma.

49. 46. ​​The cartridge of claim 45, wherein the lens is bonded to the polystyrene substrate to form a channel having a depth of 0.1 mm or less and seal the cartridge.

50. 50. The cartridge of any one of claims 44 to 49, wherein the sample is whole blood, plasma, serum, urine, CSF, saliva, gastric juice, interstitial fluid, or lymph.

51. The cartridge according to any one of claims 44 to 50, wherein the sample is an undiluted sample.

52. 52. The cartridge of any one of claims 44 to 51, wherein the cartridge is configured to measure bilirubin at equilibrium.

53. A cartridge according to any one of claims 44 to 52, wherein the cartridge is calibrated with a traceable bilirubin standard.

54. The cartridge described in claim 53, wherein the bilirubin standard is commercially available bilirubin used to calibrate a probe, the probe is used to calibrate a calibration complex, and the calibration complex is used to calibrate the cartridge.

55. one or more collection devices for collecting samples from the patient; A sensor according to any one of claims 1 to 16, or a composition according to any one of claims 17 to 21 comprising one or more probes in a suitable carrier. Kit including:

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