Human Serum Albumin Concentration Research Articles

Multilayer Heterogeneous Membrane Biosensor Based on Multiphysical Field Coupling for Human Serum Albumin Detection.

A factor closely associated with renal disease status in clinical diagnosis is abnormal human serum albumin (HSA) concentration levels in human body fluids urine, serum, etc. The surface stress biosensor was developed as a new type of biosensor to detect protein molecule concentration and has a wide range of clinical applications. However, further sensitivity improvement is required to achieve higher detection performance. Herein, MXene/PDMS/Fe3O4/PDMS of the multilayer heterogeneous membrane biosensor (MHBios) based on the coupling of the magnetic field, electric field, and surface stress field was successfully developed to achieve high sensitivity HSA detection through magnetic sensitization. The modified antibody specifically binds to HSA at the AuNP layer, allowing the biosensor to convert the surface stress caused by PDMS film deformation into an electrical signal. When the biosensor was exposed to a uniform magnetic field, the conductive path of the conductive layer was reshaped further as the magnetic force amplified the deformation of the PDMS film, enhancing the conversion of biological signals to electrical signals. The results exhibited that the detection limit (LOD) of the MHBios was 78 ng/mL when HSA concentration was 0-50 μg/mL, which was markedly lower than the minimum diagnostic limit of microalbuminuria. Furthermore, the MHBios detected HSA in actual samples, confirming the potential for early disease screening.

Influence of bovine and human serum albumin on the binding kinetics of biomolecular interactions.

Bovine serum albumin (BSA) containing buffers are the standard blocking buffer in biosensing, yet human serum is the intended application for most clinical sensors. However, the effect of human serum albumin (HSA) on binding assays remains underexplored. A simple and well-studied assay (human IgG/goat anti-human IgG) was investigated with a surface plasmon resonance (SPR) sensor to address this fundamental question in sensing. Calibrations were performed with buffers containing various concentrations of bovine or human serum albumin, as well as full and diluted bovine or IgG-depleted human serum. It was found that HSA or human serum, but not BSA or bovine serum, significantly affected the SPR shift and binding constants of the assay. Interestingly, large differences were also observed depending on whether the animal or human antibody was immobilized on the SPR chip for detection, highlighting that matrix protein/analyte/receptor interactions play a significant role in the response. We find that the interaction of soluble HSA with human IgG interferes with the recognition region, affecting the binding constant, and thus results obtained in BSA are not necessarily applicable to clinical samples or in vivo conditions. We also clearly demonstrate why a minimum dilution of 1 : 10 is often required in SPR assays to remove most background effects. Taken together, these results show that: (1) BSA does not affect the binding constant between antibodies and thus serves its purpose well when only surface blocking is intended, (2) HSA is an adequate surrogate for human serum in assay optimization, and (3) blocking buffers should be prepared with HSA in the optimization steps of assays to be translated to human blood or serum.

Association between different concentrations of human serum albumin and 28-day mortality in intensive care patients with sepsis: A propensity score matching analysis.

Background: Human serum albumin (HSA) is a commonly used medication for the treatment of sepsis. However, there is no conclusive evidence as to whether different concentrations of HSA are associated with patient prognosis. This study aimed to evaluate the association between different concentrations of HSA and 28-day mortality in patients with sepsis. Methods: The data for this retrospective study were collected from the Medical Information Mart for Intensive Care IV database. Patients with sepsis were divided into two groups according to the concentration of HSA received: 25% and 5% HSA. The primary outcome of this study was the 28-day mortality in patients with sepsis. To ensure the robustness of our findings, we used multivariate Cox regression, propensity score matching, double-robust estimation, and inverse probability weighting models. Results: A total of 76,943 patients were screened, of whom 5,009 were enrolled. 1,258 and 3,751 patients received 25% and 5% HSA, respectively. The 28-day mortality rate was 38.2% (481/1,258) for patients in the 25% HSA group and 8.7% (325/3,751) for patients in the 5% HSA group. After propensity score matching, 1,648 patients were identified. The inverse probability weighting model suggested that 5% HSA received was associated with lower 28-day mortality (hazard ratio [HR]: 0.63, 95% confidence interval [CI]: 0.54-0.73, p < 0.001). Subgroup and sensitivity analysis confirmed the robustness of the results. Conclusion: In patients with sepsis, 5% HSA received may be associated with a lower risk of 28-day mortality than 25% HSA. Further randomized controlled trials are required to confirm this association.

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.

Combined use of microbubbles of various sizes and single-transducer dual-frequency ultrasound for safe and efficient inner ear drug delivery.

We have previously applied ultrasound (US) with microbubbles (MBs) to enhance inner ear drug delivery, with most experiments conducted using single-frequency, high-power density US, and multiple treatments. In the present study, the treatment efficacy was enhanced and safety concerns were addressed using a combination of low-power-density, single-transducer, dual-frequency US (I SPTA= 213 mW/cm2) and MBs of different sizes coated with insulin-like growth factor 1 (IGF-1). This study is the first to investigate the drug-coating capacity of human serum albumin (HSA) MBs of different particle sizes and their drug delivery efficiency. The concentration of HSA was adjusted to produce different MB sizes. The drug-coating efficiency was significantly higher for large-sized MBs than for smaller MBs. In vitro Franz diffusion experiments showed that the combination of dual-frequency US and large MB size delivered the most IGF-1 (24.3± 0.47 ng/cm2) to the receptor side at the second hour of treatment. In an in vivo guinea pig experiment, the efficiency of IGF-1 delivery into the inner ear was 15.9 times greater in animals treated with the combination of dual-frequency US and large MBs (D-USMB) than in control animals treated with round window soaking (RWS). The IGF-1 delivery efficiency was 10.15 times greater with the combination of single-frequency US and large size MBs (S-USMB) than with RWS. Confocal microscopy of the cochlea showed a stronger distribution of IGF-1 in the basal turn in the D-USMB and S-USMB groups than in the RWS group. In the second and third turns, the D-USMB group showed the greatest IGF-1 distribution. Hearing assessments revealed no significant differences among the D-USMB, S-USMB, and RWS groups. In conclusion, the combination of single-transducer dual-frequency US and suitably sized MBs can significantly reduce US power density while enhancing the delivery of large molecular weight drugs, such as IGF-1, to the inner ear.

An AIE luminogen self-assembled nanoprobe for efficient monitoring of the concentration and structural transition of human serum albumin

Monitoring the content and structural transition of human serum albumin (HSA) is valuable for diagnosing hypoalbuminemia and albuminuria and elucidating its transport function, respectively. Herein, we developed a multifunctional and efficient nanoprobe based on the self-assembly of an amphiphilic aggregation-induced emission luminogen, TPNN, for HSA detection and structure monitoring. TPNN molecules formed loose self-assembly in aqueous media and emitted faint fluorescence due to vigorous intramolecular motion. In the presence of HSA, a remarkable fluorescence enhancement accompanied by dissociation of TPNN assembly was observed due to the site-specific binding of TPNN to the middle long-narrow pocket of HSA. This binding blocked the intramolecular motion while producing a sensitive, selective, fast and stable fluorescence turn-on response to HSA, making TPNN assembly suitable for the HSA assay. TPNN assemblies also showed superior selectivity to HSA than bovine serum albumin (BSA) due to the distinct binding modes, and enabled us to distinguish the two homologous proteins. Furthermore, the generated TPNN-HSA complex underwent stepwise fluorescence quenching in the presence of protein denaturants and proteases, which revealed the process and kinetics of HSA unfolding and cleavage. TPNN assembly provides a powerful tool for accurate quantification of HSA in serum and urine and real-time monitoring of HSA structural transition.

Highly Luminescent Positively Charged Quantum Dots Interacting with Proteins and Cells†

Comprehensive SummaryWe have studied interactions between positively charged MUTAB‐stabilized quantum dots (QDs) and model proteins, serum and live cells using fluorescence correlation spectroscopy (FCS), dynamic light scattering (DLS), time‐resolved photoluminescence (PL) and live‐cell fluorescence imaging. Using human serum albumin (HSA) as a model protein, we measured the growth of a protein adsorption layer (“protein corona”) via time‐resolved FCS. Corona formation was characterized by an apparent equilibrium dissociation coefficient, KD ≈ 10 μM. HSA adlayer growth was surprisingly slow (timescale ca. 30 min), in stark contrast to many similar measurements with HSA and other proteins and different NPs. Time‐resolved PL data revealed a characteristic quenching behavior depending on the QD surface coverage with HSA. Taken together, we found that MUTAB‐QDs initially bind HSA molecules weakly (KD ≈ 700 μM); however, the affinity is enhanced over time, presumably due to proton injection into the MUTAB layer by HSA triggering ligand dissociation. This process was also observed with human blood serum, showing equal kinetics for comparable HSA concentration. Moreover, imaging experiments with cultured human cells (HeLa) revealed that MUTAB‐QDs bind to the cell membrane and perforate it. This process is reduced upon pre‐adsorption of proteins on the MUTAB‐QD surfaces.

Combined microwave dielectrometry and molecular dynamic study of aqueous solutions of human serum albumin with additives

We combined the microwave dielectrometry method for the complex permittivity experimental study of aqueous Human Serum Albumin (HSA) solutions and a molecular dynamics simulation of similar modelling aqueous solutions. Using microwave dielectrometry we obtained that the real and imaginary parts of complex permittivity of all studied solutions (HSA water solutions, HSA water solutions with NaCl or glucose) decrease at the HSA concentration growing. The molecular dynamics simulation of HSA aqueous solutions showed that the increase of the HSA concentration causes the number of H-bound increase between water and protein molecules and leads to complex permittivity value decrease for the tested solutions. Thus, the molecular dynamics simulation results are in good agreement with microwave dielectrometry experimental data for HSA aqueous solutions with the addition of NaCl and glucose. Our study can be applied in the ecological investigations of wastewater samples owing to demonstrated sensitivity of complex permittivity parameters measured to the small changes of the concentration in the solutions.

Personalized diagnostic approach and indirect quantification of extravasation in human anaphylaxis.

Anaphylaxis is the most acute and life-threatening manifestation of allergic disorders. Currently, there is a need to improve its medical management and increase the understanding of its molecular mechanisms. This study aimed to quantify the extravasation underlying human anaphylactic reactions and propose new theragnostic approaches. Molecular determinations were performed in paired serum samples obtained during the acute phase and at baseline from patients presenting with hypersensitivity reactions. These were classified according to their severity as Grades 1, 2 and 3, the two latter being considered anaphylaxis. Tryptase levels were measured by ImmunoCAP, and serum protein concentration was quantified by Bradford assay. Human serum albumin (HSA) and haemoglobin beta subunit (HBB) levels were determined by Western blot and polyacrylamide gel electrophoresis, respectively. A total of 150 patients were included in the study. Of them, 112 had experienced anaphylaxis (83 and 29 with Grade 2 and 3 reactions, respectively). Tryptase diagnostic efficiency substantially improved when considering patients' baseline values (33%-54%) instead of the acute value threshold (21%). Serum protein concentration and HSA significantly decreased in anaphylaxis (p < .0001). HSA levels dropped with the severity of the reaction (6% and 15% for Grade 2 and 3 reactions, respectively). Furthermore, HBB levels increased during the acute phase of all hypersensitivity reactions (p < .0001). For the first time, the extravasation underlying human anaphylaxis has been evaluated based on the severity of the reaction using HSA and protein concentration measurements. Additionally, our findings propose new diagnostic and potential therapeutic approaches for this pathological event.

Is Protein‐Mediated Uptake Of Drugs By OATPs, Via Protein‐Lipid Interaction, A Real Phenomenon Or An Artifact?

There is considerable interest in the observation that OATP‐mediated transport is significantly increased in the presence of albumin or plasma proteins, a phenomenon called the “protein‐mediated uptake (PMU)” effect (Miyauchi et al., 2018; Liang et al., 2020; Bi et al., 2021). PMU proposes an interaction between the drug‐albumin complex and the cell surface leading to enhanced dissociation of the drug‐albumin complex resulting in increased local unbound drug concentration and therefore an “apparent” increased uptake intrinsic clearance (CLint) (Miyauchi et al., 2018; Kim et al., 2019). This PMU effect helps overcome some (but not all) of the underprediction of hepatic clearance (CL) of OATP drugs by IVIVE. If the PMU hypothesis is correct, the following should be observed in the presence vs. absence of plasma proteins: 1) the active (e.g. by OATP1B1) and passive uptake CLint of the drug should increase to the same extent; and 2) an increase in the slope (i.e. the uptake rate), but not the intercept of the uptake vs. time curve. To determine if the above is true, we determined the total, active (OATP1B1‐mediated) and passive CLint of a cocktail of OATP1B1 drugs [fluvastatin (FLV), pitavastatin (PTV), cerivastatin (CRV), atorvastatin (ATV) and rosuvastatin (RSV)], in the absence and presence of 1%, 2%, or 5% human serum albumin (HSA), by HEK293 MOCK and OATP1B1‐transfected cells at concentrations much less than their reported OATP1B1 Km values. In addition, the unbound drug concentration of each drug, in the absence and presence of HSA, was maintained approximately the same in all experiments. We found: 1) In the presence of HSA, the increase in the passive CLint(i.e. of the unbound drug) was greaterthan for either the total or active uptake CLint of the drug; and 2) in the presence of HSA, the intercept of the uptake curve increased in proportion to the HSA concentration used. An increase in the intercept is usually interpreted as non‐specific binding (NSB) of the drug to the cells/labware; in this case, NSB of the albumin‐drug complex to the cells. Therefore, we asked if PMU can be explained in whole or part by NSB of the albumin‐drug complex to cells or labware? We found that: 1) PMU on passive/active uptake of ATV, FLV and RSV was largely due to NSB (i.e. an artifact). However, it could only partially explain the effect of 5% HSA on OATP1B1‐mediated PTV uptake or the passive uptake of CRV. If the PMU of PTV is a real phenomenon, the unbound inhibitory capacity (IC50,u) of PTV towards OATP1B1 should decrease as the IC50,u is determined ONLY by the interaction between the drug and the transporter and independent of NSB or passive uptake. Indeed, the OATP1B1 IC50,u of PTV (on RSV uptake) in the presence of 5%HSA decreased by 80%, but as expected, that of ATV was unaffected. In conclusion, our results suggests that, except for PTV, the PMU of the drugs studied is likely an artifact. Therefore, mechanisms other than the presence of plasma proteins should be investigated to explain the underprediction of hepatic CL by IVIVE.Reference: Bi YA. et al. (2021) Drug Metabolism and Disposition 49:188–201. Kim SJ. et al. (2019) Drug Metabolism and Disposition 47:94–103. Liang X. et al. (2020) Drug Metabolism and Disposition 48:1283–1292. Miyauchi S. et al. (2018) Drug Metabolism and Disposition 46:259–267.

In Vitro Reactivity of the Glucose Degradation Product 3,4-Dideoxyglucosone-3-ene (3,4-DGE) towards Abundant Components of the Human Blood Circulatory System.

3,4-Dideoxyglucosone-3-ene (3,4-DGE) is a glucose degradation product present in processed foods and medicinal products. Additionally, its constant formation from 3-deoxyglucosone in plasma has been suggested. Due to its α,β-unsaturated dicarbonyl moiety, 3,4-DGE is highly reactive and has shown harmful effects in vitro. Here, we investigated the impact of major components of the human blood circulatory system on 3,4-DGE in vitro. Under physiological conditions, plasma concentrations of human serum albumin (HSA) reacted efficiently with 3,4-DGE, resulting in only 8.5% of the initial 3,4-DGE concentration after seven hours (vs. 83.4% without HSA, p < 0.001). Thereby, accessible thiol groups were reduced from 0.121 to 0.064 mol/mol HSA, whereas ketoprofen binding and esterase-like activity of HSA were not affected. Plasma concentrations of glutathione (GSH) reacted immediately and completely with 3,4-DGE, leading to two stereoisomeric adducts. Plasma concentrations of immunoglobulin G (IgG) bound to 3,4-DGE to a lower extent, resulting in 62.6% 3,4-DGE after seven hours (vs. 82.2% in the control, p < 0.01). Immobilized human collagen type IV did not alter 3,4-DGE concentrations. The results indicated that particularly HSA, GSH, and IgG readily scavenge 3,4-DGE after its appearance in the blood stream, which may be associated with a reduced antioxidative and cytoprotective activity for the living cells and, thus, the human organism by blocking free thiol groups.

Assessing the Validity of Normalizing Aflatoxin B1-Lysine Albumin Adduct Biomarker Measurements to Total Serum Albumin Concentration across Multiple Human Population Studies.

The assessment of aflatoxin B1 (AFB1) exposure using isotope-dilution liquid chromatography-mass spectrometry (LCMS) of AFB1-lysine adducts in human serum albumin (HSA) has proven to be a highly productive strategy for the biomonitoring of AFB1 exposure. To compare samples across different individuals and settings, the conventional practice has involved the normalization of raw AFB1-lysine adduct concentrations (e.g., pg/mL serum or plasma) to the total circulating HSA concentration (e.g., pg/mg HSA). It is hypothesized that this practice corrects for technical error, between-person variance in HSA synthesis or AFB1 metabolism, and other factors. However, the validity of this hypothesis has been largely unexamined by empirical analysis. The objective of this work was to test the concept that HSA normalization of AFB1-lysine adduct concentrations effectively adjusts for biological and technical variance and improves AFB1 internal dose estimates. Using data from AFB1-lysine and HSA measurements in 763 subjects, in combination with regression and Monte Carlo simulation techniques, we found that HSA accounts for essentially none of the between-person variance in HSA-normalized (R2 = 0.04) or raw AFB1-lysine measurements (R2 = 0.0001), and that HSA normalization of AFB1-lysine levels with empirical HSA values does not reduce measurement error any better than does the use of simulated data (n = 20,000). These findings were robust across diverse populations (Guatemala, China, Chile), AFB1 exposures (105 range), HSA assays (dye-binding and immunoassay), and disease states (healthy, gallstones, and gallbladder cancer). HSA normalization results in arithmetic transformation with the addition of technical error from the measurement of HSA. Combined with the added analysis time, cost, and sample consumption, these results suggest that it may be prudent to abandon the practice of normalizing adducts to HSA concentration when measuring any HSA adducts—not only AFB1-lys adducts—when using LCMS in serum/plasma.

Human Serum Albumin Dimerization Enhances the S2 Emission of Bound Cyanine IR806.

Cyanine molecules are important phototheranostic compounds given their high fluorescence yield in the near-infrared region of the spectrum. We report on the frequency and time-resolved spectroscopy of the S2 state of IR806, which demonstrates enhanced emission upon binding to the hydrophobic pocket of human serum albumin (HSA). From excitation-emission matrix spectra and electronic structure calculations, we identify the emission as one associated with a state having the polymethine chain twisted out of plane by 103°. In addition, we find that this configuration is significantly stabilized as the concentration of HSA increases. Spectroscopic changes associated with the S1 and S2 states of IR806 as a function of HSA concentration, as well as anisotropy measurements, confirm the formation of HSA dimers at concentrations greater than 10 μM. These findings imply that the longer-lived S2 state configuration can lead to more efficient phototherapy agents, and cyanine S2 spectroscopy may be a useful tool to determine the oligomerization state of HSA.

Contribution of diafiltration and adsorption to vancomycin clearance in a continuous hemodiafiltration circuit model in vitro.

Vancomycin (VCM) is eliminated mainly by diafiltration under continuous hemodiafiltration (CHDF), but the contribution of adsorption to CHDF clearance (CLCHDF ) of VCM using a polyacrylonitrile and sodium methallyl sulfonate copolymer membrane coated with polyethylenimine (AN69ST) or a polymethylmethacrylate (PMMA) membrane is unknown. This study sought to investigate the contribution of diafiltration and adsorption to the CLCHDF of VCM using AN69ST and PMMA membranes in vitro. An in vitro CHDF circuit model was developed. The initial concentration of VCM was 50μg/mL and human serum albumin (HSA) was prepared at a concentration of 0, 2.5, or 5.0g/dL. The effluent flow rate (Qe) was set at 800, 1500, or 3000mL/h. The CLCHDF , diafiltration rate, and adsorption rate of VCM were calculated. Total CLCHDF of VCM using the AN69ST membrane increased and decreased with increasing Qe and HSA concentration, respectively. Diafiltration and adsorption rates were 82.1±9.8% and 12.1±6.1% under all conditions, respectively. Total CLCHDF using the PMMA membrane increased with increasing Qe. Diafiltration and adsorption rates were 89.2±20.4% and 4.6±17.0% under all conditions, respectively. The observed CLCHDF values significantly correlated with the predicted CLCHDF , calculated according to a previous study as the product of Qe and the plasma unbound fraction. Diafiltration predominantly contributed to CLCHDF of VCM using AN69ST and PMMA membranes. When diafiltration rather than adsorption mainly contributes to the CLCHDF of VCM, the CLCHDF could be predicted from the Qe and HSA concentration, at least in vitro.

Bimodal Turn‐On Fluorescent Probe for Photophysical and Electrochemical Detection of Human Serum Albumin in Clinical Samples

AbstractA simple, easy to synthesize, cost‐effective fluorescent turn‐on sensor is developed for the selective detection of human serum albumin (HSA) in both biological and environmental samples. The biosensor is unique with qualities of rapidly quantifying HSA levels both at lower and higher concentrations in divergent bio‐fluids with high sensitivity. This probe has also been successfully used for the quantitative determination of serum albumin levels in a large number of patients’ samples having different clinical manifestations. Electrochemical detection of HSA concentrations can also be achieved with this biosensor using a potentiostat. Thus, this probe offers a unique potential of diagnosing HSA levels directly from patient samples using its bimodal (i.e., photophysical and electrochemical) properties, which are hitherto unknown to date.

Selective pathological and intracellular detection of human serum albumin by photophysical and electrochemical techniques using a FRET-based molecular probe

A simple, easily synthesizable, low-cost, fluorescent turn-on probe is presented herein for the selective and quantitative detection of human serum albumin (HSA) in different biological fluids collected from patients with various clinical manifestations. The sensor can detect HSA level by both photophysical and electrochemical means. The developed probe is also efficient in rapid quantification of HSA level in single living cell, cell lysate and tissue extract with high sensitivity. Both higher (millimolar) and trace (micromolar) amount of serum albumin can be accurately quantified using this probe in vast array of biomedical samples. This chemical sensor is also used as a part of Förster Resonance Energy Transfer (FRET) based system adding further accuracy to the measurement technique. Intracellular concentrations of HSA can be measured as well as imaged using this newly synthesized probe. Electrochemical detection of HSA concentrations can also be achieved with this biosensor using a potentiostat. Thus, this probe offers a unique potential of diagnosing HSA levels directly in various biological samples, using its bimodal (i.e., photophysical and electrochemical) properties which is hitherto unknown till date.

Rapid monitoring of serum albumin as a biomarker of liver and kidney diseases using femtosecond laser-induced fluorescence

Albumin is the most abundant serum protein that transports hormones, free fatty acids, bilirubin, various ions, and drugs. The current study investigated the potential application of laser-induced fluorescence (LIF) in clinical analysis of human serum albumin (HSA) as a biomarker of liver and kidney disease. The excitation wavelength and HSA concentrations were systematically varied, and the LIF spectrum was recorded, in order to construct a standard calibration curve. Over a wide range of excitation wavelengths, excitation at 350 nm showedthe maximum fluorescence emission centered at 500 nm. We compared the determination of certain concentrations of HSA using both LIF and conventional laboratory assays. The LIF technique proved to be highly accurate and efficient. It may be concluded that femtosecond LIF provides a new, easy, very sensitive, precise, and direct method of detecting albumin in various biological samples.

A silicon nitride ion sensitive field effect transistor‐based immunosensor for determination of urinary albumin

AbstractThe development of an immunosensor‐based biosensor to detect the human serum albumin (HSA) was developed as a low‐cost and label‐free electrical detection through silicon nitride ion‐sensitive field‐effect transistor (Si3N4‐ISFET). This sensor was functionalized using 3‐Aminopropyltriethoxysilane (APTES). The silanized Si3N4‐ISFET surface was covalently linked with antibody against HSA (Anti‐HSA) via glutaraldehyde (GA) to function as an HSA immunosensor for detection of HSA. The interaction between HSA and Anti‐HSA on this device is monitored by changing of the gate potential at a constant drain current of 50 μA. The relationship between HSA concentration and the gate potential change of HSA immunosensor is linear up to 500 μg/ml with a detection limit of 5 μg/ml. This HSA immunosensor offers a good precision in both intra‐ and inter‐assay with a relative standard deviation of 6.61%, 7.48%, 10.69%, and 9.67%, 13.16%, 15.52% for 100, 200, and 400 μg/ml. The average percentage of recovery assay for the concentration of 32.1, 102.1, and 202.1 μg/ml is 96.55%. This sensor also provides its satisfactory selectivity when testing with Bence Jones protein solution. In addition, the results that was done by this HSA immunosensor is similar to the conventional method using immunoturbidimetric detection with a linear regression of 0.99; n = 40 with a paired sample t‐test of &lt;0.01. Thus, this HSA immunosensor could be applied to urinary microalbumin detection because of its simplicity and satisfactory correlation value.

Exploring the Molecular Level Interaction of Human Serum Albumin with Calcium Oxalate Monohydrate Crystals.

Human serum albumin (HSA) is one of the most abundant proteins in the blood plasma, urine as well as in the organic matrix of renal calculi. Macromolecules present in the urine modulate kidney stone formation either by stimulating or inhibiting the crystallization process. In the present study, the effect of HSA protein on the growth of calcium oxalate monohydrate crystal (COM) was investigated. Crystal growth assay was used to measure oxalate depletion in the crystal seeded solution in the presence of HSA. HSA concentrations exhibiting effect on crystal growth were selected for FTIR and XRD analysis. In silico docking was performed on seven different binding sites of HSA. Albumin plays dual role in the growth of calcium oxalate crystallization. FTIR and XRD studies further revealed HSA exerted strain over crystal thus affecting its structure by interacting with amino acids of its pocket 1. Docking results indicate that out of 7 binding pocket in protein, calcium oxalate interacts with Arg-186 and Lys-190 amino acids of pocket 1. Our study confirms the role of HSA in calcium oxalate crystallization where acidic amino acids arginine and lysine bind to COM crystals, revealing molecular interaction of macromolecule and crystal in urolithiasis.

A fluorescent sensor recognized by the FA1 site for highly sensitive detection of HSA

Human serum albumin (HSA), as the most abundant protein in blood plasma, plays a crucial role in many physiological processes. The abnormal HSA level in serum or in urine is often associated with various diseases. Therefore, to achieve highly sensitive and selective quantification of HSA is of great importance for disease diagnosis and preventive medicine. Herein, an HSA-selective light-up fluorescent sensor, DCM-ML, was successfully developed for quantitative detection of HSA. DCM-ML exhibited good (photo-) stability and strong fluorescence enhancement around 630 nm in the presence of HSA in complex samples containing numerous biological analytes. Upon addition of HSA into DCM-ML containing solution, a good linear relationship (R2 > 0.99) between the fluorescence intensity of DCM-ML and HSA concentration from 0 to 0.08 mg/mL was obtained with the detection limit of 0.25 μg/mL. The sensing mechanism of the sensor towards HSA was demonstrated to be via recognition in the fatty acid site 1 (FA1), instead of the most reported binding sites (Sudlow I and II) in HSA, for the first time, by both the displacement experiments and molecular docking simulation. Thus, DCM-ML can also be assumed as a potential FA1 site-binding marker for examining drugs binding to the FA1 site in HSA. At last, the utilization of sensor DCM-ML for quantification and validation of HSA in urine samples and cell culture medium was effectively demonstrated. Therefore, the development of DCM-ML should find great application potentials in the fields of analytical chemistry and clinical medicine as a highly sensitive HSA sensor.