Bilirubin In Human Serum Research Articles

Selective and sensitive detection of bilirubin by Fe3+-mediated porphyrin red fluorescence "on-off-on" sensing platform

Bilirubin (BR) serves as a crucial clinical marker for jaundice and liver dysfunction, making its quantitative detection amidst numerous biomolecular interferences of paramount importance. In this work, 5, 10, 15, 20-tetrakis(4-methoxycarbonylphenyl)porphyrin (TPPCOOMe) was utilized as the fluorophore, and Fe3+ was used as the mediator to achieve quantitative detection of BR in human serum through the "on-off-on" fluorescence signal change. The non-fluorescent metalloporphyrin complex TPPCOOMe@Fe3+ generated by the interaction between Fe3+ and TPPCOOMe served as the fluorescence sensing platform. With the addition of BR, the good binding ability between BR and Fe3+ released TPPCOOMe into the sensing medium and restored red fluorescence, while creating conditions for the redox reaction between BR and Fe3+. The fluorescence probe exhibited a good linear relationship with BR in the concentration range of 0–8 μM, the detection limit was as low as 14 nM and the response time was 2 min. Furthermore, a test paper strip-based sensor coupled with a smartphone color-read App was employed to test the fluorescence color changes of TPPCOOMe@Fe3+ for BR sensing, and this intelligent sensing platform had been successfully achieved for the actual detection of BR in human serum without the influence of other metal ions and important biomolecules.

Efficient detection of bilirubin in human serum through a displacement approach

The displacement of bilirubin in human serum with THYQ provides fluorescence “turn on” detection for bilirubin, which is in good agreement with the clinical results.

Turn-off near-infrared fluorescent probe for free bilirubin detection constructed by enhanced excimer emission

In this study, an amphiphilic near-infrared fluorescent molecule (denoted BCPB) was employed as a fluorescent probe to detect free bilirubin. In an aqueous solution, the micellar assemblies of BCPB possess a strong excimer emission at 660 nm, which was dramatically quenched upon the addition of bilirubin. It has been proven that fluorescence quenching is mainly attributed to photoinduced electron transfer (PET) from BCPB to bilirubin. As a fluorescent probe of bilirubin, BCPB showed advantages, such as fast response (<1 min), good anti-interference ability, and low limit of detection (0.33 μmol L−1, S/N = 3). BCPB was successfully applied to detect free bilirubin in human serum and urine, and the detection showed very high accuracy.

A Low-Cost Paper-Based Device for the Colorimetric Quantification of Bilirubin in Serum Using Smartphone Technology.

Total bilirubin values have been used as a potential marker to pre-screen and diagnose various liver-based diseases such as jaundice, bile obstruction, liver cancer, etc. A device known as KromaHealth Kit, composed of paper and an acrylic backbone, is developed to quantify total bilirubin in human serum using image processing and machine learning technology. The biochemical assays are deposited on absorbent paper pads that act as reaction zones when serum is added. A dedicated smartphone app captures images of the colorimetric changes on the pad and converts them into quantitative values of bilirubin. The range of bilirubin concentration that can be quantified using the device ranges from 0.5 mg/dl to 7.0 mg/dl. The precision, limit of detection, interference analysis, linearity, stability, and comparison with a predicate are studied in this paper in accordance with clinical and laboratory standards institute. The results indicate that the KromaHealth Kit can be used as an inexpensive alternative to conventional bilirubin testing in clinical settings. With its level of precision, ease-of-use, long shelf-life, and short turnaround time, it will prove to be invaluable in limited-resource settings.

A selective and sensitive fluorescent probe for bilirubin in human serum based on europium(III) post-functionalized Zr(IV)-Based MOFs

In the present study, a kind of Eu(III) post-functionalized Zr(IV)-based metal-organic framework (UiO-66(COOH)2, Zr-MOF: Eu3+) was synthesized and utilized as an independently luminescent probe for sensing bilirubin (BR) in human serum, a biomarker of jaundice hepatitis. It can be served as a turn-off fluorescent switch for BR because its red emission from Eu3+ can be easily quenched by BR through a fluorescent resonant energy transfer (FRET) process between BR and its ligands, and as a result, BR is recognized successfully. Particularly, Zr-MOF: Eu3+ has shown many appealing properties, such as high sensitivity, quick response (less than 1 min), broad response window (0–15 μM), and excellent selectivity. Most importantly, a kind of portable test paper based on Zr-MOF: Eu3+ probe has been developed for directly assessing the level of BR in real human serum and further diagnosing bilirubin-related diseases via visually observing the luminescent color variation.

Water-soluble MoS2 quantum dots as effective fluorescence probe for the determination of bilirubin in human fluids

Bilirubin is an important biomarker in the diagnosis and prognosis of patients with liver disorders. Herein, we report a simple, rapid, sensitive and selective quantitative determination of bilirubin using molybdenum disulfide quantum dots (MoS2 QDs) as a probe. The MoS2 QDs were synthesized through a hydrothermal route by using sodium molybdate and cysteine as the starting materials. The obtained MoS2 QDs exhibits strong luminescence property and excellent stability. The HR-TEM image shows that the size of the prepared MoS2 QDs was 2.4 nm with a spherical morphology. The MoS2 QDs emit intense blue photoluminescence (with excitation/emission peaks at 310/392 nm) under UV light and the fluorescence of MoS2 QDs was drastically quenched by the addition of bilirubin. The Förster resonance energy transfer (FRET) and inner filter effect (IFE) between MoS2 QDs and bilirubin resulted in the fluorescence quenching of MoS2 QDs. The present method demonstrated high sensitivity towards bilirubin with the limit of detection (LOD) of 2.1 nM (S/N = 3). The MoS2 QDs probe showed remarkable selectivity to bilirubin over other possible interferences. Moreover, the present fluorophore was successfully utilized for the detection of bilirubin in human serum and urine samples. QDs based fluorescence probe for the recognition of bilirubin is reported for the first time.

Selective non-enzymatic total bilirubin detection in serum using europium complexes with different β-diketone-derived ligands as luminescence probes.

Three europium(III) complexes, Eu(ectfd)3 (Hectfd = 1-(9-ethyl-9H-carbazol-7-yl)-4,4,4-trifluorobutane-1,3-dione), Eu(tta)3 (Htta = 4,4,4-trifluoro-1-(thiophen-2-yl)-butane-1,3-dione), and Eu(dbt)3 (Hdbt = 2-(4',4',4'-trifluoro-1',3'-dioxobutyl)dibenzothiophene), were synthesized and employed to detect total bilirubin (BR) in blood-serum samples. UV-visible absorption and fluorescence (FL) spectroscopies were used to evaluate the selectivity of each europium (III) fluorescence probe to BR, which was shown to remarkably reduce the luminescence intensities of the europium(III) complexes at a wavelength of 612 nm. The luminescence intensity of each complex is linearly related to BR concentration. Eu(tta)3 was shown to be the more-appropriate fluorescence probe for the sensitive and reliable detection of total BR in blood serum samples than either Eu(ectfd)3 or Eu(dbt)3. This observation can be ascribed to special σ-hole bonding between Htta and BR. In addition, the optimal pH test conditions for the detection of BR in human serum by the Eu(tta)3 probe were determined. Sensitivity was shown to be dramatically affected by the pH of the medium. The experimental results reveal that pH 7.5 is optimal for this probe, which coincides with the pH of human serum. Furthermore, BR detection using the Eu(tta)3 luminescence probe is simple, practical, and relatively free of interference from coexisting substances; it has a minimum detection limit (DL) of 68 nM and is a potential candidate for the routine assessment of total BR in serum samples. Graphical Abstract ᅟ.

Application of high-performance liquid chromatography combined with ultra-sensitive thermal lens spectrometric detection for simultaneous biliverdin and bilirubin assessment at trace levels in human serum

We present the applicability of a new ultra-sensitive analytical method for the simultaneous determination of biliverdin and bilirubin in human serum. The method comprises isocratic reversed-phase (RP) C18 high-performance liquid chromatography (HPLC) and thermal lens spectrometric detection (TLS) based on excitation by a krypton laser emission line at 407nm. This method enables the separation of IX-α biliverdin and IX-α bilirubin in 11min with limit of detection (LOD) and limit of quantitation (LOQ) for biliverdin of 1.2nM and 3nM, and 1nM and 2.8nM for bilirubin, respectively. In addition, a step-gradient elution was set up, by changing the mobile phase composition, in order to further enhance the sensitivity for bilirubin determination with LOD and LOQ of 0.5nM and 1.5nM, respectively. In parallel, an isocratic HPLC-DAD method was developed for benchmarking against HPLC-TLS methods. The LOD and LOQ for biliverdin were 6nM and 18nM, and 2.5nM and 8nM for bilirubin, respectively. Additionally, both isocratic methods were applied for measuring biliverdin and free bilirubin in human serum samples (from 2 male and 2 female healthy donors). Combining isocratic HPLC method with TLS detector was crucial for first ever biliverdin determination in serum together with simultaneous free bilirubin determination. We showed for the first time the concentration ratio of free bilirubin versus unbound biliverdin in human serum samples.

Selective and Sensitive Sensing of Free Bilirubin in Human Serum Using Water-Soluble Polyfluorene as Fluorescent Probe

The adherence of serum protein on conjugated polymer is a major bottleneck in the application of the latter for selective sensing of small biomolecules in blood serum. In this report, we present new polyfluorenes with d-glucuronic acid appendage that is a nonreceptor for any serum protein, thereby providing a platform for selective sensing of free bilirubin in the clinically relevant range of 50 μmol/L in human blood serum. The appended d-glucuronic acid formed noncovalent interactions with bilirubin, which in conjunction with favorable spectral overlap between the polymers and bilirubin facilitated efficient FRET process in aqueous solutions. Addition of bilirubin resulted in the quenching of the polyfluorene emission with simultaneous appearance of bilirubin emission exhibiting visual emission color change from blue to light green. The polymer remained stable in serum even under severe basic conditions and exhibited high selectivity with visual sensitivity only toward free bilirubin in human ser...

Simple and sensitive method for the quantification of total bilirubin in human serum using 3-methyl-2-benzothiazolinone hydrazone hydrochloride as a chromogenic probe

We here describe a new spectrophotometric method for measuring total bilirubin in serum. The method is based on the cleavage of bilirubin giving formaldehyde which further reacts with diazotized 3-methyl-2-benzothiazolinone hydrazone hydrochloride giving blue colored solution with maximum absorbance at 630 nm. Sensitivity of the developed method was compared with Jendrassik-Grof assay procedure and its applicability has been tested with human serum samples. Good correlation was attained between both methods giving slope of 0.994, intercept 0.015, and R 2 = 0.997. Beers law obeyed in the range of 0.068–17.2 μM with good linearity, absorbance y = 0.044 C bil + 0.003. Relative standard deviation was 0.006872, within day precision ranged 0.3–1.2% and day-to-day precision ranged 1–6%. Recovery of the method varied from 97 to 102%. The proposed method has higher sensitivity with less interference. The obtained product was extracted and was spectrally characterized for structural confirmation with FT-IR, 1H NMR.

Laboratory performance in neonatal bilirubin testing using commutable specimens: a progress report on a College of American Pathologists study.

Abstract Context.—In 2003 the Chemistry Resource Committee of the College of American Pathologists introduced a commutable specimen in the Neonatal Bilirubin Surveys. This specimen was intended to help evaluate all bilirubin methods. Objective.—To evaluate the effect of commutable specimens on the performance of selected clinical analyzers in measuring neonatal bilirubin from 2003 through 2006. Design.—A human serum–based specimen enriched with unconjugated bilirubin in human serum has been included since 2003 in the Neonatal Bilirubin Surveys. The bilirubin values of these specimens were determined by the reference method and used to evaluate results reported by various chemistry analyzers. Results.—Coefficients of variation for College of American Pathologists All Data ranged from 4.9% to 6.2% for the Neonatal Bilirubin Survey. However, coefficients of variation for the 4 major instrument groups (Dimension, Olympus, Synchron, and Vitros), which report 65% of all results, varied from 2% to 3%. College of...

Laboratory Performance in Neonatal Bilirubin Testing Using Commutable Specimens: A Progress Report on a College of American Pathologists Study

In 2003 the Chemistry Resource Committee of the College of American Pathologists introduced a commutable specimen in the Neonatal Bilirubin Surveys. This specimen was intended to help evaluate all bilirubin methods. To evaluate the effect of commutable specimens on the performance of selected clinical analyzers in measuring neonatal bilirubin from 2003 through 2006. A human serum-based specimen enriched with unconjugated bilirubin in human serum has been included since 2003 in the Neonatal Bilirubin Surveys. The bilirubin values of these specimens were determined by the reference method and used to evaluate results reported by various chemistry analyzers. Coefficients of variation for College of American Pathologists All Data ranged from 4.9% to 6.2% for the Neonatal Bilirubin Survey. However, coefficients of variation for the 4 major instrument groups (Dimension, Olympus, Synchron, and Vitros), which report 65% of all results, varied from 2% to 3%. College of American Pathologists All Data mean bilirubin values were within 0.46 mg/dL (7.8 micromol/L) of the reference method mean in 2003; in subsequent years these differences became larger, peaking at 1.87 mg/dL (32 micromol/L) in 2005. The large systematic error of bilirubin measurements is due primarily to failure of instrument manufacturers to produce reliable bilirubin calibrators. Primary calibrators should consist of human serum enriched with unconjugated bilirubin. Bilirubin values must be assigned by the reference method, the performance and robustness of which are reported in this article. Secondary calibrators distributed to users must be traceable to primary calibrators.

Determination of total bilirubin in human serum by chemiluminescence from the reaction of bilirubin and peroxynitrite

Peroxynitrous acid (ONOOH) was produced by the on-line reaction of acidified hydrogen peroxide with nitrite in a flow system, and peroxynitrite (ONOO−) was generated from ONOOH in NaOH solution. A weak chemiluminescent (CL) emission was observed due to the production of singlet oxygen (O21) during the decomposition of ONOO−. Bilirubin and its conjugate were found to enhance the CL emission of O21 in a suitable micellar medium. For the first time, the feasibility of employing the present CL system for the sensitive and selective determination of total bilirubin contents in human serum was demonstrated and the results were compared with certified values. The present method showed a great improvement on overcoming bis(2,4,6-trichlorophenyl)oxalate CL highly insolubility in aqueous solution and exhibiting higher tolerance towards interferences than redox reaction of bilirubin with various oxidizing agents such as sodium hypochlorite and iodine. The recoveries of bilirubin were found to fall in the range between 95 and 108%. The detection limits (S/N=3) for bilirubin and its conjugate were determined to be 10 and 8ngml−1, respectively. The relative standard deviations (R.S.D.) for the consecutive CL detection of a series of bilirubin (30μgl−1) and bilirubin ditaurite (25μgl−1) were 3.2 and 2.9% (n=11), respectively.

第16回日本総合健診医学会学術大会講演抄録 酵素的測定法による血清総ビリルビンの正常値の検討

第16回日本総合健診医学会学術大会講演抄録 酵素的測定法による血清総ビリルビンの正常値の検討

Azodipyrroles of unconjugated and conjugated bilirubin using diazotized ethyl anthranilate in dimethyl sulfoxide

We have developed a diazotization technique in which both conjugated and unconjugated bilirubin react completely. The method represents a crucial modification of the ethyl anthranilate diazo reaction originally described by K. P. M. Heirwegh, J. Fevery, J. A. T. P. Meuwissen, and J. de Groote (1974, Methods Biochem. Anal. 22, 205–250). In the presence of dimethyl sulfoxide (2 ml/ml of sample and diazo reagent), conjugated and unconjugated bilirubin in human serum and human, rat, and mouse bile reacted rapidly and completely. The azopigments were stable for at least 4 h. Addition of human serum to unconjugated bilirubin, bilirubin monoglucuronide, and human bile did not influence azopigment formation. Because the reaction solution was optically clear, total azopigments could be measured by spectrophotometry or separated and quantitated by high-performance liquid chromatography without prior extraction into nonpolar solvents. Alternatively, the pigments could also be extracted into 2-pentanone for analysis by thin-layer or high-performance liquid chromatography. This method allows the quantitation of total bilirubin and analysis of individual ethyl anthranilate azopigments after a single diazotization step.

Determination of unconjugated bilirubin in human serum by optical rotation measurements

Determination of unconjugated bilirubin in human serum by optical rotation measurements

A new method for the determination of unconjugated bilirubin in human serum by spectropolarimetry

A new spectropolarimetric method for the quantitative determination of unconjugated bilirubin in blood plasma is devised. It makes use of the very strong binding of unconjugated bilirubin to serum albumin and the development of the extrinsic Cotton effects as a consequence of the binding. When using circular dichroism, the ellipticity at 407 nm is directly proportional to the bilirubin concentration up to a level of about 30 mg per 100 ml. The method is not affected by haemolysis or the presence of aromatic phenols, amines or sulphonamides. High levels of conjugated bilirubin, however, give too high ellipticity values. The spectropolarimetric method is statistically compared with a direct-reading bilirubinometer method.

Automated Procedure for Determination of Bilirubin in Human Sera

Abstract An automated procedure is presented, based on the method of Bruchner [Amer. J. Clin. Pathol. 32, 513 (1959)], for determination of both total and conjugated bilirubin. The method is standardized by use of bilirubin in human serum, which is stabilized by a mixture of formamide and cyanide. Hemoglobin does not interfere at concentrations less than 500 mg/100 ml, and the effect of lipemia is minimized.

A reference for determination of bilirubin concentration in serum using bilirubin in cyanide-formamide for enrichment of serum.

Unconjugated bilirubin is highly soluble in formamide with cyanide (0.10 mol/1), giving a solution stable at 25 °C in the dark for 4 hours. Human serum (at pH = 9.5) can be enriched with this solution to at least 430 μmol/1 (250 mg/1) even when the volume fraction of formamide is below 0.02. The bilirubin complex in the ensuing preparation (employing infant serum) seems to have the same stability and spectrophotometric properties as in serum from hyperbilirubinaemic infants. This reference preparation is suitable for standardization of methods using azobilirubin or direct spectro-photometry for the determination of total bilirubin in human serum.

Quantitative separation and determination of bilirubin and conjugated bilirubin in human serum

A simple method—suitable for use in the clinical chemical laboratory—is described for the determination of bilirubin and conjugated bilirubin in human serum, the pigments being separated before the quantitative determination. The reproducibility of the method is about 0.10 mg of pigment per 100 ml of serum. The presence of conjugated bilirubin can be visually established from about 0.15 mg/100 ml upwards. No interference is caused by serum turbidity, or by moderately haemolytic sera. Deviating azo colours such as those which occasionally occur in other determinations, were not observed. The method can be used for determinations in small quantities of serum (e.g. 0.04 ml), so that neonatal blood can also be examined.