Quantification Of Human Serum Albumin Research Articles

1,4-Dihydropyridine-based FA1 site-specific fluorescent probes for the selective detection and quantification of HSA levels in biofluids.

Human serum albumin (HSA) is a multifunctional circulatory protein essential for many physiological processes including oncotic pressure maintenance, ligand/drug binding and transport, antioxidant activity, etc. Abnormal HSA levels in biological fluids have been reported in a variety of clinical disorders, making it a potential biomarker for early diagnosis. Low serum albumin levels have been linked to increased long- and short-term mortality rates in ICU patients. Therefore, quantifying HSA in biofluids such as serum and urine offers a convenient approach for the early identification of underlying clinical conditions and assessing the risk factors. Herein, we report a series of fluorescent 1,4-dihydropyridine (DHP) derivatives for the detection and quantification of HSA in biofluids. Their response towards HSA can be tuned by varying the substituents at the C-4 and the N-1 of the DHP ring. Depending on the nature of the substituents, they generated either a turn-on or ratiometric response with a LoD in low nanomolar or subnanomolar levels. A pair of enantiomers obtained by introducing a chiral center on the N-substituents highlighted the importance of stereochemistry in HSA-ligand interactions. Quantification of HSA in complex biofluids, such as blood serum and urine, was also accomplished using these probes. The high selectivity of some of the probes towards HSA over the homologous BSA allowed the discrimination of these two proteins. The preferred binding location of the probes was the hemin binding site and the detection mechanism was identified as the restriction of intramolecular rotation. Additionally, a prototype of a smartphone-integrated point-of-care device was also fabricated to demonstrate the feasibility of utilizing these probes in clinical settings.

Quantification of human serum albumin by combining chymotrypsin/trypsin digestion coupled with LC-MS/MS technique

The quantification of albumin is important in clinical medicine because the concentration of albumin in biological fluids is closely related to human health. In this study, we developed a highly selective and robust assay to determine human serum albumin (HSA) in human plasma by combining chymotrypsin/trypsin digestion coupled with targeted LC-MS/MS technique. Human plasma samples were denatured, reduced, alkylated, and digested with both chymotrypsin and trypsin to generate surrogate peptides. A unique chymotryptic peptide (NAETF) arising from human serum albumin was finally selected for targeted LC-MS/MS detection and quantification. Numerous parameters related to the targeted LC-MS/MS assay were evaluated, including lower limit of quantitation (LLOQ), linearity range, enzyme digestion efficiency, accuracy and precision. The LC-MS/MS assay was linear in the concentration range 0.05–1 mg/mL with intra-day and inter-day precision <10.2% and accuracy ranging from −3.94% to 4.89%. The assay was successfully applied to determine HSA in 148 human plasma samples.

LESS is more: Achieving sensitive protein detection by combining electric field effects and surface-enhanced Raman scattering

We present a novel and effective sensing method that combines surface-enhanced Raman scattering (SERS) with electric fields for achieving sensitive and label-free detection of proteins in physiologically relevant media. Using as an example the detection and quantification of human serum albumin (HSA) and creatinine in artificial urine, we combine experiments and simulations to demonstrate how properly applied electric field effects facilitate accelerated transport and deterministic capture of proteins and silver nanoparticles onto the SERS-active substrate. Our experimental approach yields a sandwich assay that amplifies protein concentration and creates higher surface density of hotspots on SERS-active substrates. We call our method “LESS” for brevity, an acronym that captures its key attributes: Label-free, Electric field-assisted, Sandwich-based, and Surface-enhanced Raman scattering. LESS was compared with a standard SERS assay and was shown to produce over 10-fold signal enhancement. Moreover, it offers protein detection and identification even in cases where the standard SERS assay produces no results. Reproducible SERS signals and detection thresholds well below the physiological levels of these proteins in urine were achieved. When combined with chemometric analysis, LESS allows us to extract diagnostic information by classifying SERS spectra acquired from solutions of the individual proteins or their mixtures.

A novel 1,8-naphthalimide-based fluorescent chemosensor for the detection of HSA in living cells.

Human serum albumin (HSA) is an essential protein for maintaining human health. Accurate detection and quantification of HSA are of great significance for disease diagnosis and biochemical research. Here, a new HSA fluorescent probe BNPE based on the 1,8-naphthalimide fluorophore was designed and synthesized. The probe could recognize HSA through a twisted intramolecular charge transfer mechanism, effectively avoid the interference of most substances, and realize HSA fluorescence imaging in living cells.

Serum Albumin Inspired Self-Assembly/Disassembly of a Fluorogenic Nanoprobe for Real-Time Monitoring and Quantification of Urinary Albumin with Live Cell Imaging Application.

Abnormal levels (high/low) of urinary human serum albumin (HSA) are associated with a number of diseases and thus act as an essential biomarker for quick therapeutic monitoring and biomedical diagnosis, entailing the urgent development of an effective chemosensor to quantify the albumin levels. Herein, we have rationally designed and developed a small fluorogenic molecular probe, (Z)-2-(5-((8-hydroxy-2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]quinolin-9-yl) methylene)-4-oxo-2-thioxothiazolidin-3-yl) acetic acid (HJRA) with a twisted intramolecular charge transfer (TICT) property, which can easily self-assemble into nonfluorescent nanoaggregates in aqueous solution. However, HJRA nanoaggregates can selectively bind with serum albumin proteins (HSA/BSA) in ∼100% PBS medium, thereby facilitating the disassembly of nanoaggregates into monomers, exhibiting a clear turn-on red fluorescent response toward HSA and BSA. Analysis of the specific binding mechanism between HJRA and HSA using a site-selective fluorescence displacement assay and molecular docking simulations indicates that a variety of noncovalent interactions are responsible for the disassembly of nanoaggregates with the concomitant trapping of the HJRA monomer at site I in HSA, yielding a substantial red emission caused by the inhibition of intramolecular rotation of HJRA probe inside the hydrophobic cavity of HSA. The limit of detection (LOD) determined by the 3σ/slope method was found to be 1.13 nM, which is substantially below the normal HSA concentration level in healthy urine, signifying the very high sensitivity of the probe toward HSA. The comparable results and quick response toward quantification of HSA in urine by HJRA with respect to the Bradford method clearly point toward the superiority of this method compared to the existing ones and may lead to biomedical applications for HSA quantification in urine. It may also find potential application in live-cell imaging of HSA.

Rapid Point-of-Care Quantification of Human Serum Albumin in Urine Based on Ratiometric Fluorescence Signaling Driven by Intramolecular H-Bonding.

Human serum albumin exerts multifunctions, such as maintaining the oncotic pressure of plasma, carrying hydrophobic molecules, and acting as the most important antioxidant in the blood. Lower serum albumin levels are linked to several cardiovascular diseases, and dysfunction of albumin reabsorption in the kidney is linked to liver disease, renal disorder, and diabetes. Albumin is thus a powerful diagnostic and prognostic marker; however, its quantification in urine by readily affordable tools is challenging owing to its very low concentration. To address this issue, we developed a ratiometric fluorescent probe with multiple advantages through a systematic structure variation of a benzocoumarin fluorophore and, further, a prototype of a smartphone-based point-of-care device. We determined albumin levels in urine and observed that a smoking person has notably higher urine albumin than a nonsmoking person. The cheap device provides a promising tool for albumin-associated disease diagnosis in communities with limited resources.

Time-resolved fluorescence determination of albumin using ZnGeO:Mn luminescence nanorods modified with polydopamine nanoparticles.

A novel time-resolved fluorescence (TRF) pobe is constructed to detect human serum albumin (HSA) by exploiting ZnGeO:Mn persistent luminescence nanorods (ZnGeO:Mn PLNRs) and polydopamine nanoparticles (PDA NPs). HSA-induced dynamic quenching leads to the fluorescence decrease of ZnGeO:Mn PLNRs, providing the basis for quantitative analysis of HSA. The excellent photo-thermal conversion performance of PDA NPs is helpful to the collision process between ZnGeO:Mn PLNRs and HSA, inducing significant improvement of sensitivity. HSA is quantified by measuring time-resolved fluorescence at 540nm under excitation of 250-nm light. Under optimal conditions, HSA in the linear range 0.1-100ngmL-1 are detected by this PDA-mediated ZnGeO:Mn probe with high sensitivity and selectivity, and the detection limit is 36pgmL-1 (3σ/s). The RSD for the quantification of HSA (5ngmL-1, n = 11) is 5.2%. The practicability of this TRF probe is confirmed by accurate monitoring HSA contents in urine samples, giving rise to satisfactory spiking recoveries of 96.2-106.0%.

Fluorescence Enhancement Based Quantification of Human Serum Albumin from Biological Sample Using Indole Based Nanosuspension: Molecular Interactions and Molecular Docking Studies.

Fluorescent 3-[(E)-(2-phenylhydrazinylidene) methyl]-1H-indole (PHI) was synthesized by condensation of indole-3-carboxaldehyde and phenyl hydrazine in presence of acetic acid and ethanol and after spectral characterization used further to prepare its aqueous nano suspension by reprecipitation method using polyvinylpyrrolidone (PVP) as stabilizer. The average particle size of nano suspension measured by Dynamic Light Scattering (DLS) was found 77.5nm while FESEM microphotograph showed spherical morphology. The blue shift in the absorption spectrum and stokes shifted fluorescence of nanosuspension of PHI compared to its monomer spectrum in dilute solution indicate formation of H-type aggregate by face to face overlapping of the molecules.The aggregation induced enhanced emission (AIEE) of PVP capped nanosuspension of PHI is increased appreciably by presence of aqueous solution of human serum albumin (HSA). A suitable mechanism of molecular binding interactions based on complex formation between PHI nanoaggregate and HSA through PVP is proposed. Fluorescence life time, zeta potential and particle size data of PHI nanoparticles (PHINPs) obtained in presence of different amounts of HSA are in support of molecular interactions leading to complex formation. The molecular docking studies showed that HSA and PVP capped PHINPs exhibit strong hydrogen bonding interaction. The fluorescence enhancement effect induced in PHI nanosuspension is used further to develop analytical method for quantitative estimation of HSA in aqueous biological sample solution.

A flavonoid-based fluorescent probe enables the accurate quantification of human serum albumin by minimizing the interference from blood lipids.

We herein provide a simple design strategy to improve the sensing specificity towards human serum albumin by incorporating a nitrobenzene quencher into a traditional polarity-sensitive probe in responding to the interference from blood lipids.

Differential recognition and quantification of HSA and BSA based on two red-NIR fluorescent probes

It is always a challenge to achieve the selective recognition between human serum albumin (HSA) and bovine serum albumin (BSA) because of their highly homologous primary structure. In this work, we reported two red-NIR fluorescence probes, BI-FPI and NTPS-FPI, for HSA and BSA differential recognition. BI-FPI showed remarkable emission enhancement toward HSA over BSA, while NTPS-FPI exhibited the opposite selectivity toward BSA. The obvious different interaction for probes with HSA and BSA were demonstrated by site specific competitive binding experiments and molecular docking studies. BI-FPI located within site I of HSA, which mainly depended on the hydrophobic residues and π-π interaction. While NTPS-FPI docked into the interface between subdomains II and IIIA of BSA, and the strong hydrogen bond interaction was the main contributor for the binding. Furthermore, the good calibration graphs of BI-FPI to HSA enabled us to develop the attractive multiple functional probe for qualitatively and quantitatively detecting HSA, and the detection limit (0.01μM, 0.66mg/L) met the requirements of traditional HSA assay in serum or in urine (30mg/L).

An environment-sensitive fluorescent probe for quantification of human serum albumin: Design, sensing mechanism, and its application in clinical diagnosis of hypoalbuminemia

Unusual human serum albumin concentration in serum or urine represents an important early biomarker for various medical conditions and diseases. Therefore, accurate quantification of HSA concentration in real biological samples has gained great importance for clinical diagnosis and preventive medicine. However, most of previous reports focus on detection of serum samples collected from healthy individuals, rather than the patients with abnormal serum albumin level. Further validation of HSA probes in clinical diagnosis is urgently needed yet remains challenging. Here, we reported an environment-sensitive fluorescent probe CD1, which exhibited fast response, high sensitivity, and excellent selectivity towards HSA. The limit of detection was determined to be as low as 8.6 nM (0.57 mg/L). Based on this method, the quantitative determination of HSA in real serum samples from both healthy individuals and patients with hypoalbuminemia are firstly reported, which demonstrated its practical application in clinical disease pre-diagnosis.

A red-NIR emissive probe for the selective detection of albumin in urine samples and live cells.

A dicyanomethylene-4H-chromene-derived turn-on fluorescence probe (TG-SA) has been designed and synthesised for the selective detection of human serum albumin (HSA). TG-SA exhibits good photostability and selective red-near infrared (NIR) fluorescence enhancement in the presence of HSA in complex samples containing numerous biological analytes. The probe predominantly binds domain II of HSA that accounts for the observed selective fluorescence enhancement. The red-NIR fluorescence probe TG-SA can be effectively used for staining HSA/SA in gel electrophoresis, quantification and validation of HSA in clinical urine samples and monitoring serum albumin in live cells.

Flow-injection with enhanced evaporative light scattering detector detection and quantification of human serum albumin using gold nanoparticles

The ELSD response for HSA was enhanced by using GNPs, making it possible to determine HSA by ELSD.

Self-assembled near-infrared dye nanoparticles as a selective protein sensor by activation of a dormant fluorophore.

Design of selective sensors for a specific analyte in blood serum, which contains a large number of proteins, small molecules, and ions, is important in clinical diagnostics. While metal and polymeric nanoparticle conjugates have been used as sensors, small molecular assemblies have rarely been exploited for the selective sensing of a protein in blood serum. Herein we demonstrate how a nonspecific small molecular fluorescent dye can be empowered to form a selective protein sensor as illustrated with a thiol-sensitive near-IR squaraine (Sq) dye (λabs= 670 nm, λem= 700 nm). The dye self-assembles to form nonfluorescent nanoparticles (Dh = 200 nm) which selectively respond to human serum albumin (HSA) in the presence of other thiol-containing molecules and proteins by triggering a green fluorescence. This selective response of the dye nanoparticles allowed detection and quantification of HSA in blood serum with a sensitivity limit of 3 nM. Notably, the Sq dye in solution state is nonselective and responds to any thiol-containing proteins and small molecules. The sensing mechanism involves HSA specific controlled disassembly of the Sq nanoparticles to the molecular dye by a noncovalent binding process and its subsequent reaction with the thiol moiety of the protein, triggering the green emission of a dormant fluorophore present in the dye. This study demonstrates the power of a self-assembled small molecular fluorophore for protein sensing and is a simple chemical tool for the clinical diagnosis of blood serum.

Portable-Surface Plasmon Resonance Biosensor Immunoassays for the Human Serum Albumin Detection

The screening of urine human serum albumin (HSA) is significant for the early diagnosis and intervention of the nephropathia. This study developed sensitive HSA detection assay with a self-made portable-SPR biosensor based on the reflection light intensity interrogation. Anti-HSA monoclonal antibody was immobilized onto the gold sensing area and a directive assay for HSA was developed. A detection range of 1–100μg/mL HSA was found. The described label-free, real-time immunoassay for the quantification of HSA in urine is a rapid, sensitive, alternative method available for the clinic detection.

Practical applications of high-affinity, albumin-binding proteins from a group G streptococcal isolate

Binding proteins that have high affinities for mammalian plasma proteins that are expressed on the surface of bacteria have proven valuable for the purification and detection of several biologically important molecules from human and animal plasma or serum. In this study, we have isolated a high affinity albumin-binding molecule from a group G streptococcal isolate of bovine origin and have demonstrated that the isolated protein can be biotinylated without loss of binding activity and can be used as a tracer for quantification of human serum albumin (HSA). The binding protein can be immobilized and used as a selective capture reagent in a competitive ELISA format using a biotinylated HSA tracer. In this assay format, the sensitivity of detection for 50% inhibition of binding of HSA was less than 1 microg/ml. When attached to the bacterial surface, this binding protein can be used to deplete albumin from human plasma, as analyzed by surface-enhanced laser desorption ionization time of flight mass spectrometry.

Development of Humanized Mice for the Study of Hepatitis C Virus Infection

The development of a small animal model for hepatitis C virus (HCV) infection is a critical issue for the development of novel anti-HCV drugs. To this aim, we have tried many different approaches for generating mice carrying humanized liver. Main efforts were focused on the transplantation of human hepatocytes into immunocompromised mice (SCID−/−, Bg−/−) carrying a genetic lethal liver disease (Alb-uPA). Survival of homozygotic animals should largely depend on early transplantation with healthy hepatocytes. In parallel to establishing a colony of Alb-uPA/SCID/Bg mice, we developed a microsurgical procedure for intrasplenic xenotransplantation of healthy hepatocytes in 1-week-old mice. So far, we generated several chimeras by xenotransplanting human hepatocytes in Alb-uPA+/+/SCID−/−/Bg−/− mice at 1 week after birth. In a first step, identification of successfully engrafted animals is possible by quantification of human serum albumin and human alpha 1 antitrypsin in mouse sera. Additional preliminary histomorphological analysis of liver sections from chimeric animals was also carried out. One of the mice was transiently infected with HCV, reaching viremia levels of ∼10 5 genomes/mL. However, the efficiency of this system to generate chimeric mice is still very limited. We are currently exploring the use of more robust models of hepatic disease. Moreover, we have been also exploring novel strategies for the generation of chimeric mice by xenotransplanting human adult stem cells, instead of human hepatocytes, at preimmune stages of development.

Direct quantification of human serum albumin in human blood serum without separation of γ-globulin by the total internal reflected resonance light scattering of thorium–sodium dodecylbenzene sulfonate at water/tetrachloromethane interface

A direct quantification of human serum albumin (HSA) in blood serum samples without separation is proposed based on the measurements of total internal reflected resonance light scattering (TIR-RLS) at water/tetrachloromethane (H 2O/CCl 4) interfaces. In the pH range of 6.37–6.59, the coadsorption of the binary complex of HSA–Th(IV) with sodium dodecylbenzene sulfonate occurs at the H 2O/CCl 4 interface, forming an amphiphilic layer and displaying greatly enhanced TIR-RLS signals with the maximum peak located at 340–370 nm. The enhanced TIR-RLS intensity is in proportion to the HSA concentration in the range 0.15–1.0 μg ml −1 . The limit of detection is 14.4 ng ml −1 . The contents of HSA in blood serum samples were determined with the recovery of 97.1–102.3% and RSD of 0.6–2.9%, which are identical to those obtained according to the spectrofluorimetric method using chrome azurol S.