Selective human serum albumin sensor from the screening of a fluorescent rosamine library

A fluorescent dye library approach for the development of a bioanalyte sensor was sought. The screening of a rosamine dye library against diverse macromolecules led to the discovery of a highly sensitive human serum albumin binder, G13, with approximately 36-fold fluorescence intensity change. G13 showed a highly selective response to HSA over other macromolecules including albumins from other species. The potential use of G13 for the detection of HSA in biofluids is described.     

Fluorogenic Detection of Human Serum Albumin Using Curcumin-Capped Mesoporous Silica Nanoparticles

Mesoporous silica nanoparticles loaded with rhodamine B and capped with curcumin are used for the selective and sensitive fluorogenic detection of human serum albumin (HSA). The sensing mesoporous silica nanoparticles are loaded with rhodamine B, decorated with aminopropyl moieties and capped with curcumin. The nanoparticles selectively release the rhodamine B cargo in the presence of HSA. A limit of detection for HSA of 0.1 mg/mL in PBS (pH 7.4)-acetonitrile 95:5 v/v was found, and the sensing nanoparticles were used to detect HSA in spiked synthetic urine samples.     

Towards biosensors based on conducting polymer nanowires

We report the electrochemical deposition of poly(pyrrolepropylic acid) nanowires, their covalent modification with antibodies and their conversion into potential functional sensor devices. The nanowires and the devices were characterised by optical microscopy, fluorescence microscopy, electron microscopy and electrical measurements. Fluorescence images, current–voltage (I–V) profiles and real-time sensing measurements demonstrated a rapid and highly sensitive and selective detection of human serum albumin (HSA), a substance that has been used to diagnose incipient renal disease. The detection is based on the selective binding of HSA onto anti-HSA that is covalently attached to the nanowires. The binding changes the electrical properties of the nanowires thus enabling the real-time detection. Whilst the utility of the research was demonstrated for protein binding/detection, the technology could easily be designed for the detection of other analytes by the modification of polymer nanowires with other analyte-specific molecules/biomolecules. Therefore, the technology has the potential to positively impact broad analytical applications in the biomedical, environmental and other sectors. Figure Real-time dynamic current response on sequential exposure of buffer, bovine serum albumin (BSA) and human serum albumin (HSA) onto anti-HSA modified poly (pyrrolepropylic acid) nanowires. Fluorescence images of poly(pyrrolepropylic acid) nanowire (top right) and polypyrrole nanowire control (bottom right) after sequential treatment with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), anti HSA and fluorophore-labeled HSA.     

Highly sensitive spectrophotometric determination of human serum albumin with 3',4',5',6'-tetrachlorogallein-molybdenum(VI) complex

A highly sensitive spectrophotometric determination of human serum albumin (HSA) with 3',4',5',6'-tetrachlorogallein (T.Cl.Gall)-Mo(VI) complex in a Triton X-100 + polyvinyl alcohol micellar medium is proposed. This method can be used to determine up to ca. 150 micrograms/10 ml of HSA from the optical absorbance at 640 nm, and is superior in sensitivity to the other extremely sensitive spectrophotometric methods. The great sensitivity of this method results from the use of third-derivative spectrophotometry. The binding parameters of T.Cl.Gall-Mo(VI) complex to HSA are n = 77.3 and K = 1.05 x 10(4) M-1 as determined from dual double-reciprocal plots. It is suggested that the colored complex in this system may be the association complex between [HSA]m+ and [MoVI(T.Cl.Gall)2]n- involving hydrophobic interaction between HSA and T.Cl.Gall. The proposed method should also be useful for the detection and determination of some peptides (e.g. low molecular weight peptides containing basic amino acids), as well as proteins.     

Fluorimetric study of the interaction between human serum albumin and quinolones-terbium complex and its application

A highly sensitive spectrofluorimetric method is proposed for determination of human serum albumin (HSA) and some quinolone drugs. Using quinolones-terbium (Tb3+) complex as a fluorescent probe, in the buffer solution of pH 7.8, HSA can remarkably enhance the fluorescence intensity of the quinolones-Tb3+ complex at 545 nm and the enhanced fluorescence intensity of Tb3+ ion is in proportion to the concentration of HSA and quinolone drugs. Optimum conditions for the determination of HSA were also investigated. The linear ranges and limits of detection are 8.0 x 10(-9) to 8.0 x 10(-8) mol L(-1), 4.20 x 10(-9) mol L(-1) (for HSA); 1.0 x 10(-6) to 4.0 x 10(-6) mol L(-1), 1.87 x 10(-8) mol L(-1) (for norfloxacin) and 1.0 x 10(-7) to 1.0 x 10(-6) mol L(-1), 4.82 x 10(-8) mol L(-1) (for enoxacine), respectively. This method is simple, practical and relatively free interference from coexisting substances, as well as much more sensitive than most of the existing assays.     

Electrochemical Study on the Interaction of Protein with Bromothymol Blue and Its Analytical Application

The interaction of bromothymol blue（BB） with human serum albumin（HSA） was studied by electrochemical techniques and a sensitive method for proteins assay was developed. When BB interacted with HSA, the voltammetric peak current value of BB decreased linearly with the concentration of HSA in a range of 1.0--40.0 mg/L, and the peak potential shifted negatively. Based on the results, a sensitive assay method for proteins, such as HSA, bovine serum albumin（BSA）, and egg albumin etc. was established. This method was further applied to determining the HSA in healthy human blood samples, and the results are not significantly different from those obtained by the classic Coomassie Brilliant Blue G-250 spectrophotometic method. The detecting conditions of this method were optimized and the interaction mechanism was discussed. The results show that the electrochemical parameters（formal potential E^0, standard rate constant of the electrode reaction ks, parameter of kinetic nα） of BB have no obvious changes before and after the interaction, which indicate that BB can interact with HSA, forming an electrochemical non-active complex. The equilibrium constant（βs） and the binding ratio（m） for this complex were calculated. The m is 4 and βs is 1.41 × 10^19. This method is fast, simple, highly sensitive, and has good selectivity, which can be used in clinical measurements.     

A novel chemiluminescence technique for quantitative measurement of low concentration human serum albumin.

An efficient and highly sensitive chemiluminescence (CL) technique is proposed in the current study for detection of low levels of human serum albumin (HSA). Chemiluminescence (CL) produced during interaction between fluoresceinyl cypridina luciferin analog (FCLA)-1O2 can be modified with the presence of HSA. The conventional CL technique uses a quenching effect of HSA for its quantitative measurement. We are reporting here that the CL intensity can be enhanced, rather than quenched, by the addition of HSA. The CL signal can be linearly correlated with the HSA concentration over a clinically interesting range of 5 x 10(-9) - 8 x 10(-8) mol L(-1), with a detection limit of 2.5 x 10(-9) mol L(-1). The determination result was consistent with that obtained from conventional methods. One possible mechanism of HSA detection technique using CL enhancement approach is discussed. Intermolecular energy transfer in chemiluminescence systems and changes of microenvironment are likely to be contributors of the CL enhancement with HSA.     

Motion‐Induced Changes in Emission as an Effective Strategy for the Ratiometric Probing of Human Serum Albumin and Trypsin in Biological Fluids

Herein, we report the formation of a highly luminescent, pH‐sensitive, thermoreversible nanoaggregate in pure aqueous medium through the self‐agglomeration of carbazole‐based amphiphiles. The self‐assembly process restricted the intramolecular motion of the molecules and induced a change in its emission signal from blue to cyan, owing to an aggregation‐induced emission (AIE) effect. A similar type of ratiometric response was also observed in the presence of human serum albumin (HSA). However, in this case, the molecular motion of the flexible fluorescent probe was restricted by its embedded microenvironment, owing to a motion‐induced change in emission (MICE) effect, not by aggregation. Moreover, the probe showed quite high selectivity for HSA over other serum albumin proteins. Our carbazole‐based fluorescent probes are a unique example of the ratiometric sensing of HSA through the sole involvement of reversible noncovalent interactions. Considering the important of HSA in clinical diagnosis, a wide range of biological fluids, such as human urine, saliva, and plasma, were screened to analyze their HSA content. In addition, this system was also employed for the detection of trypsin at subnanomolar concentrations through the digestion of HSA.     

A High Sensitivity Detection Method of Singlet Oxygen and Superoxide Anion

This paper, for the first time, reports a method that can be used as a highly sensitive probe for singlet oxygen (^1O2) and superoxide anion (O2^-) in vitro or in vivo. FCLA(3,7-dihydro-6-{4-[2-(N‘-(5-fluoresceinyl)thioureido)ethoxy]phenyl}-2-methylimidazo{1,2-a}pyrazin-3-one sodium salt), a chemiluminescence (CL) analysis reagent, has been reported to sensitively react with ^1O2 and O2^- to emit photons with a spectral peak of 525nm. In this work,when human serum albumin (HSA) was added into FCLA solution to enhance the CL intensity,approximately 20 times, compared to that without HSA. The enhanced CL had the same 525 nm spectral peak, identical to that without HSA. By gradually reducing the molecular oxygen content in the solution, we find that the auto-oxidation of oxygen molecules dissolved in the solution plays an important role in the CL process. Based on these experimental evidences, we propose a novel and highly sensitive detection method of ^1O2 and O2^- which may have a great potential in chemical and medical applications.     

Direct fluorimetric determination of gamma-globulin in human serum with organic nanoparticle biosensor

This paper describes the development of organic fluorescence nanoparticles. The nanoparticles have a narrow, tunable, symmetric emission spectrum and a broad, continuous excitation spectrum. The nanoparticles have high room-temperature fluorescence quantum yields and long fluorescence lifetime. They are also photochemically stable and water-soluble. They were used as fluorescence biosensor in the determination of proteins, which was proved to be a simple, rapid and specific method. In comparison with single organic fluorephores, these nanoparticles are brighter, more stable against photobleaching, and do not suffer from blinking. Under optimal conditions, the linear ranges of the calibration curves were 0.1-4.5 microg ml(-1) for human serum albumin (HSA), 0.2-3.5 microg ml(-1) for bovine serum albumin (BSA) and 0.04-0.8 microg ml(-1) for gamma globulin (gamma-IgG), respectively. The detection limits were 0.062 microg ml(-1) for HSA, 0.036 microg ml(-1) for BSA and 0.022 microg ml(-1) for gamma-IgG, respectively. However, when the content of HSA is lower than 0.8 microg ml(-1), HSA makes little contribution to the fluorescence quenching. So, the method was applied to direct selective quantification of gamma-IgG in human blood serum without separation of HSA. The results were in good agreement with these reported by the hospital, indicating that the method presented here is not only sensitive, selective and simple, but also reliable and suitable for practical applications.     

Spectrophotometric determination of urinary protein with o-sulfophenylfluorone-metal complex.

A simple and sensitive spectrophotometric method for the determination of human serum albumin (HSA) was established based on the ternary complex-formation reaction of HSA with o-sulfophenylfluorone (SPF) as a xanthene dye and metal ion (niobium(V) and bismuth(III)) in the presence of a dispersion agent. This new method enabled the determination of HSA in the range of 1 - 15 microg/ml HSA by measuring the difference of the absorbance at 530 nm between HSA-SPF-metal ion and SPF-metal ion solutions. In the determination of HSA, this method is about 2-times more sensitive than the Pyrogallol Red-molybdenum(VI) method (PR method), which accounts for more than 80% of the quantification methods for urinary protein assays in Japan. There was no significant difference between the results obtained by the present method and the PR method for human urine samples. The binding process between the SPF-metal complex and HSA was studied by determining the binding parameters and the thermodynamic parameters.     

Study of Hemoglobin and Human Serum Albumin Glycation with Electrochemical Techniques

High concentration glucose in diabetes mellitus may stimulate nonenzymatic glycation of proteins. Hemoglobin (Hb) and human serum albumin (HSA) are among the most sensitive proteins for the modification by glucose. In this paper, we report our study of Hb and HSA modification by glucose using electrochemical methods. Compared with native Hb, it is found that highly glycated Hb presents lower electron transfer reactivity and electrocatalytic activity toward O₂ and H₂O₂, and the glycation is glucose concentration and time dependent. Meanwhile, the changes of the electrochemical signal of heme after binding with HSA and glycated HSA have also suggested that proteins modified by high concentration glucose lasting for months and years in diabetes mellitus might be the reason for diabetes mellitus complication.     

Determination of proteins at nanogram levels with Bordeaux red based on the enhancement of resonance light scattering

A simple, sensitive and selective method was proposed for the determination of proteins by using a resonance light scattering technique. The weak resonance light scattering (RLS) of Bordeaux red (BR) can be enhanced greatly in the pH range 3.87-3.96 by the addition of micro amounts of proteins, resulting in four characteristic peaks in the wavelength range 250-600 nm. At the maximal wavelength of 363 nm, the enhanced RLS is proportional to the concentration of proteins in the range 0.12-10.8 microg ml-1 for bovine serum albumin (BSA) and 0.24-18.0 microg ml-1 for human serum albumin (HSA). The detection limits were 40.0 and 52.9 ng ml-1 for BSA and HSA, respectively. The present method has been applied to the determination of total proteins in human serum, urine and saliva samples. The obtained results are in good agreement with those obtained by the Bradford method with relative standard deviations (R.S.D.) of 0.9-2.3%.     

Fractionation of human plasma proteins by hydrophobic interaction membrane chromatography

This paper discusses the fractionation of human plasma proteins HSA and HIgG by hydrophobic interaction membrane chromatography. A type of microporous polyvinylidine fluoride (PVDF) membrane having 0.1 μm pore size was identified as being suitable for carrying out this separation. This membrane bound HIgG at 1.5 M ammonium sulphate concentration, a condition at which HSA did not. Based on this selective binding resulting from the selective pressure induced by the high anti-chaotropic salt concentration, these human plasma proteins were fractionated. The HIgG binding capacity of the PVDF membrane examined in this study was 42.8 mg/ml at a feed concentration of 0.45 mg/ml. Separation of simulated HSA/HIgG mixtures were carried out in the pulse and step input modes and the HSA and HIgG fractions thus obtained were analysed for purity using affinity chromatography and SDS-PAGE. HSA and HIgG purities were typically in excess of 97–98%.     

Selective Determination of Nanogram γ-Globulin in Human Blood Serum by Resonance Light Scattering of Functionalized Nano-PbS

Functionalized nano-PbS has been prepared and characterized. The functionalized nanoparticles have good dispersibility in water. Reaction of functionalized nano-PbS with γ-globulin (γ-IgG) results an enhanced resonance light scattering (RLS) around 385nm.However, when the content of HSA is lower than 0.5μg/ml^-1 the RLS enhancement is very weak and is nonlinear to concentration of HSA. Based on these results, a new direct quantitative determination method for γ-globulin in blood serum samples without separation is established.Under optimal conditions, the enhanced RLS intensity is in proportion to the γ-IgG concentration in the range 10-500ng/mL. The limit of detection is 2.75ng/mL. This method is proved to be very sensitive, rapid, simple and selective for detection of γ-IgG in blood serum.     

Effects of the Interaction of Rifamycin SV with Serum Albumins on the Resonance Rayleigh Scattering Spectra and Their Analytical Application

In pH 4.5–4.8 Britton‐Robinson buffer solution, rifamycin SV (i.e. rifamycin sodium) can react with serum albumin such as human serum albumin (HSA) and bovine serum albumin (BSA) to form macromolecular complexes by electrostatic attraction and hydrophobic force. As a result, the resonance Rayleigh scattering (RRS) of the drug was enhanced remarkably and the RRS peaks were at 374 and 552 nm. The enhancement of RRS (ΔI) is directly proportional to the concentration of HSA or BSA. The linear ranges and the detection limits are 0.03–6.0 µg/mL and 9.0 ng/mL for HSA, and 0.01–8.0 µg/mL and 2.0 ng/mL for BSA, respectively. In this work, a sensitive, selective, simple and fast method for the determination of trace amounts of serum albumin by RRS technique has been developed, which was applied to the determination of serum albumin in the synthesized samples and human urine samples with satisfactory results.     

Selective extraction of peptides in acidic human plasma by porous silica nanoparticles for peptidome analysis with 2-D LC-MS/MS

In this study, an improved method for human plasma peptidome analysis including selective porous silica nanoparticles (MCM-41) extraction and subsequent online 2-D nano-LC-MS/MS analysis was established. Enhanced enrichment efficiency for the MCM-41 extraction was obtained by adjusting the pH of the plasma sample to 2.5. A total of 1680 unique peptides were identified in the plasma sample obtained from one healthy donor, which is nearly twice the amount identified from the native state of the plasma sample. The hydrophobic property, molecular weight (MW), and pI distribution of the identified peptides at pH 2.5 and native state of the plasma sample were systematically investigated and compared. Furthermore, many unusual cleaved peptides from plasma proteins (e. g., HSA) were observed at pH 2.5, which clearly show a ladder pattern. The cleavage patterns for all of the identified peptides at pH 2.5 were summarized, and chymosin and cathepsin D were confirmed as the possible peptidases responsible for the change of cleavage pattern in peptide profiling.     

A novel TICT-based near-infrared fluorescent probe for light-up sensing and imaging of human serum albumin in real samples

Human serum albumin (HSA) has emerged as a pivotal biomarker and prognostic indicator for various human diseases. Real-time sensing and visual tracking of HSA in plasma or other biological systems will immensely facilitate the basic researchers and clinicians to better understand HSA-associated biological processes. Herein, a novel near-infrared (NIR) fluorescent probe (7-HTCF) was rationally constructed for light-up sensing and in-situ imaging of HSA in real samples, based on the principle of twisted intramolecular charge transfer (TICT). Under physiological conditions, 7-HTCF could be efficiently trapped by HSA to form a stable complex via binding on a non-drug binding site, while the complex emitted strong fluoresce signals around 670 nm. Further investigations demonstrated that 7-HTCF displayed a great combination of excellent selectivity and good chemical stability, as well as rapid fluorescent response and ultra-high sensitivity for HSA detection. Particularly, the newly developed light-up probe has been successfully utilized for quantitative detection of HSA in diluted plasma samples, while its readouts are hardly affected by the addition of therapeutic agents and herbal medicines. 7-HTCF is also successfully used for in-situ imaging of the reabsorbed HSA in living renal cells, while this dye exhibits good cell permeability and high resolution for in-situ imaging in living cells. Collectively, a novel TICT-based near-infrared fluorescent probe was devised for highly selective and ultra-sensitive sensing of HSA in plasma samples or imaging HSA in living cells, which offered a practical tool for clinical tests and for exploring HSA-associated biological processes.     

Induced self-assembly and Förster resonance energy transfer studies of alkynylplatinum(II) terpyridine complex through interaction with water-soluble poly(phenylene ethynylene sulfonate) and the proof-of-principle demonstration of this two-component ensemble for selective label-free detection of human serum albumin

The interaction of conjugated polyelectrolyte, PPE-SO(3)(-), with platinum(II) complexes, [Pt(tpy)(C≡CC(6)H(4)-CH(2)NMe(3)-4)](OTf)(2) (1) and [Pt(tpy)(C≡C-CH(2)NMe(3))](OTf)(2) (2), has been studied by UV-vis, and steady-state and time-resolved emission spectroscopy. A unique FRET from PPE-SO(3)(-) to the aggregated complex 1 on the polymer chain with Pt···Pt interaction has been demonstrated, resulting in the growth of triplet metal-metal-to-ligand charge transfer ((3)MMLCT) emission in the near-infrared (NIR) region. This two-component ensemble has been employed in a "proof-of-principle" concept for the sensitive and selective label-free detection of human serum albumin (HSA) by the emission spectral changes in the visible and in the NIR region. The spectral changes have been ascribed to the disassembly of the polymer-metal complex aggregates upon the binding of PPE-SO(3)(-) to HSA, which is rich in arginine residues and hydrophobic patches, leading to the decrease in FRET from PPE-SO(3)(-) to the aggregated platinum(II) complex. The ensemble is found to have high selectivity toward HSA over a number of polyelectrolytes, proteins and small amino acids. This has been suggested to be a result of the extra stabilization gained from the Pt···Pt and π-π interactions in addition to the electrostatic and hydrophobic interactions found in the polymer-metal complex aggregates.     

Identification of specific protein carbonylation sites in model oxidations of human serum albumin

Human serum albumin (HSA) was subjected to oxidative stress and the locations of the resulting protein carbonyls were determined using mass spectrometry in conjunction with a hydrazide labeling scheme. To model oxidative stress, HSA samples were subjected to metal-catalyzed oxidation (MCO) conditions or treated with hypochlorous acid (HOCl). Oxidation led to the conversion of lysine residues to 2-aminoadipic semi-aldehyde residues, which were subsequently labeled with biotin hydrazide. Analysis of the tryptic peptides from the samples indicates that the oxidations are highly selective. Under MCO conditions, only two of the 59 lysine residues appeared to be modified (Lys-97 and Lys-186). With HOCl, five different lysine modification sites were identified (Lys-130, Lys-257, Lys-438, Lys-499, and Lys-598). These results strongly suggest that the preferred site of modification is dependent on the nature of the oxidant and that the process relies on specific structural motifs in the protein to direct the oxidation. The high selectivity seen here provides insights into the factors that in vivo drive the selective carbonylation of specific proteins in systems under oxidative stress.