Human Serum Albumin Concentration Research Articles

The influence of culture medium and protein supplementation on the sensitivity of the Mouse Embryo Assay to detect Triton X-100

Research question: To what extent does the type and concentration of protein and the type of culture medium affect the sensitivity of Mouse Embryo Assays (MEAs) to detect Triton X-100 (TX-100) in culture media?Design: The effect of bovine (BSA) and human serum albumin (HSA) concentration was assessed by supplementing media with 0.5 or 5mg/ml. KSOM and HTF were used as complex and simple-formulation media, respectively. Variables were combined forming study groups where embryos were cultured in tested media spiked with a sublethal TX-100 concentration. The conditions of greatest sensitivity were determined by statistically comparing blastocyst formation rates and total cell counts between groups.Results: Although all the study groups showed an equal capacity of sustaining a proper embryo development, the reported sensitivity of the MEA differed between groups when subjected to TX-100. HTF significantly conferred greater sensitivity than KSOM regardless of the amount/type of protein used, whereas medium supplementation with 5mg/ml BSA rather than 0.5mg/ml also resulted in significantly higher sensitivity regardless of the type of medium used. This increase in concentration also resulted in a higher sensitivity when supplementing HTF with HSA. BSA groups provided more sensitivity than its HSA-counterparts except for the group 0.5mg/ml-KSOM. Cell counting analysis did not provide further significant conclusions.Conclusions: For TX-100 detection within culture medium, the concentration and type of protein supplementation and the culture medium type impact directly on MEA sensitivity. These results could help to standardize MEA protocols and increase their ability to detect sublethal amounts of embryotoxic substances, especially TX-100, avoiding possible clinical harmful effects.

Molecularly imprinted electrochemiluminescence sensor based on a novel luminol derivative for detection of human serum albumin via click reaction.

Anovel luminol derivative of N-(1,4-dioxo-1,2,3,4-tetrahydrophthalazin-5-yl)acrylamide (DTA) with excellent luminescence efficiency was designed and synthesized. Furthermore, a molecularly imprinted electrochemiluminescence sensor (MIECLS) was fabricated to detect ultratrace levels of human serum albumin (HSA) with high sensitivity and selectivity via a click reaction. The molecularly imprinted polymers (MIPs) were formed on the electrode surface via electropolymerization with HSA as a template molecule and catechol as a monomer. In the detection process, the -SH group of HSA on the electrode and the C = C bond of acryloyl group in DTA formed a new C-S bond via the Michael addition reaction to construct the MIECLS. The higher the concentration of HSA, the greater electrochemiluminescence (ECL) intensity measured. Taking advantage of MIECLS for ECL detection (scanning potential, - 0.4 to 0.5V), there was a good linear relationship between ECL intensity and the logarithm of HSA concentration in the range 5 × 10-9 to 1 × 10-13mgmL-1. The limit of detection (LOD) of the sensor was 1.05 × 10-15mgmL-1. The sensor exhibited outstanding selectivity and stability. Thesensor was applied to detect HSA in human serum with good recoveries of 97.7-105.2%. Theconcentration of HSA was detected by electrochemical method using the gating effect of MIP.

Chiral separation of propranolol by electrokinetic chromatography using nanodiamonds and human serum albumin as a pseudo-stationary phase in river water.

Propranolol is currently considered as an emerging contaminant in water bodies. In this study, R- and S-propranolol were determined in river samples by electrokinetic chromatography (EKC) using nanodiamonds (NDs) and human serum albumin (HSA) as a pseudo-stationary phase in order to achieve enantioseparation. Previously, river samples were preconcentrated using a column filled with Amberlite® IR-120 and Dowex® 50WX8 resins. The setting up of influential factors such as temperature, voltage, pH, and HSA and NDs concentration is accurately described along this manuscript. A multivariate study and optimization was carried out to obtain the enantioseparation of propranolol (Rs = 2.91), which was reached under the following experimental conditions: voltage of 16 kV, temperature of 16°C, phosphate buffer pH 9.5, NDs of 0.20%, and HSA of 15 μmol l-1 . The recoveries of analytes under optimal conditions were higher than 98%. The limits of detection were 0.85 μg l-1 for R- and S-propranolol. The method was applied to real samples, and the obtained results in three different water sources studied were 1.02, 0.59, and 0.30 μg l-1 for the R-enantiomer and 0.99, 0.54, and 0.28 μg l-1 for the S-enantiomer. The accuracy of the proposed methodology (including bias and precision) has allowed us to propose it as a successful tool for the control of water quality.

Exploring weak ligand-protein interactions by relaxometry of long-lived spin order.

Relaxation times of nuclear spins often serve as a valuable source of information on the dynamics of various biochemical processes. Measuring relaxation as a function of the external magnetic field turned out to be extremely useful for the studies of weak ligand-protein interactions. We demonstrate that observing the relaxation of the long-lived spin order instead of longitudinal magnetization extends the capability of this approach. We studied the field-dependent relaxation of the longitudinal magnetization and the singlet order (SO) of methylene protons in alanine-glycine dipeptide and citrate in the presence of human serum albumin (HSA). As a result, SO relaxation proved to be more sensitive to ligand-protein interaction, providing higher relaxation contrast for various HSA concentrations. To assess the parameters of the binding process in more details, we utilized a simple analytical relaxation model to fit the experimental field dependences for both SO and T1 relaxation. We also tested the validity of our approach in the experiments with trimethylsilylpropanoic acid (TSP) used as a competitor in ligand binding with HSA.

Numerical modeling of metal-dielectric metasurface as an element of microwave sensors for biomedical applications

This paper reports the results of numerical modeling of wave reflection coefficient behavior of the metal-dielectric metasurface at microwaves that can be used for biomedical applications. The study includes optimization of the working parameters of the metasurface-based structure as a sensitive element of a microwave sensoring systems for determination of proteins concentration in different solutions. In the current research a unit with the geometry, which is similar to the geometry of one well of the standard 96-multiwell laboratory microplate, is used as a resonant metasurface unit cell and liquid-holding structure. Human serum albumin (HSA) is used as a protein specimen in our model study. The present numerical modeling is based on the results of our previous experimental measurements of complex permittivity values of HSA water solutions and its biochemical reaction mixtures using microwave dielectrometry method and the developed setup. The unit cell approach calculations are performed by COMSOL Multiphysics software. The optimization of working parameters of the metal-dielectric metasurface structure with tested solutions allow us to observe the resonance effects of the wave reflection coefficient in the microwave range. We can determine the HSA concentration changes in water solutions and enzymatic reaction mixtures by the resonance frequency shift of the wave reflection coefficient of the metal-dielectric metasurface. Developed metal-dielectric metasurface-based structure demonstrates prospects to be used as a sensitive element of microwave sensors for proteins concentration determination with biomedical purposes.

A microenvironment-sensitive red emissive probe with a large Stokes shift for specific recognition and quantification of serum albumin in complex biofluids and live cells.

Human serum albumin (HSA) is regarded as a useful biomarker for rapid medical diagnosis of various disorders mainly related to the kidneys and liver. Hence, it is crucial to identify and monitor the HSA level in complex biofluids (urine and blood samples) using a simple approach. Herein, we have designed and synthesized an intramolecular charge transfer (ICT) based environment-sensitive fluorescent molecular probe, (E)-2-(3-(2-(5-methoxy-1H-indol-3-yl)vinyl)-5,5-dimethylcyclohex-2-en-1-ylidene)malononitrile (DCI-MIN), that can selectively interact with HSA in PBS buffer solution and exhibit a ∼78-fold enhancement in fluorescence intensity with a significant Stokes shift (∼126 nm), which is important to avoid interference from the excitation light. The significant red fluorescence response can be attributed to the suppression of free intramolecular rotation of the DCI-MIN probe inside the hydrophobic binding cavity of HSA and the low polar microenvironment present within HSA. According to the 3σ/slope method, the detection limit was found to be 1.01 nM (0.0671 mg L-1) in aqueous solutions, which is significantly lower than the normal level of HSA in healthy urine and blood serum, indicating its high sensitivity. DCI-MIN has the ability to exhibit useful applications, including the detection and quantification of HSA concentration in complex biofluids (human urine and blood samples) as well as the imaging of serum albumin in living cells.

A Narrative Literature Review of the Established Safety of Human Serum Albumin Use as a Stabilizer in Aesthetic Botulinum Toxin Formulations Compared to Alternatives.

Despite more than 80 years of use in a number of conditions, including in critically ill patients, comments have recently arisen regarding the safety and efficacy of human serum albumin (HSA) as a therapeutic product and stabilizer/excipient in botulinum neurotoxins. This review summarizes the literature on the safety of HSA. Beyond decades of safe use, the largest clinical dataset of HSA safety is a large meta-analysis of HSA supplier data, which found only an extremely remote risk of serious adverse events across millions of doses of therapeutic concentrations of HSA. There is a paucity of literature identifying HSA-specific adverse events when used as a stabilizer/excipient; however, studies of HSA-containing botulinum neurotoxins (BoNTs) suggest that adverse events are not related to HSA. Polysorbates, which are synthetically produced and not physiologically inert, are contained in pending or new-to-market BoNT formulations. In contrast to HSA, evidence exists to suggest that polysorbates (particularly PS20/PS80) can cause serious adverse events (e.g., hypersensitivity, anaphylaxis, and immunogenicity).

Role of albumin Cys34 redox state in the progression of differentiated thyroid carcinoma and induction of ferroptosis

Differentiated thyroid cancer (DTC) is the most prevalent endocrine malignancy worldwide and requires effective prognostic markers and therapeutic targets to optimize patient outcomes. This study investigated the potential of human serum albumin (HSA) cysteine-34 (Cys34) redox state as a prognostic indicator and therapeutic avenue for DTC. A retrospective cohort study of 99 patients with DTC undergoing radioactive iodine therapy found that higher concentrations of HSA with the reduced form of Cys34 (i.e., human mercaptalbumin [HMA]) were associated with improved progression-free survival in metastatic DTC. In vitro experiments using a DTC cell line revealed that HMA induced cytotoxic effects by triggering ferroptosis, characterized by lipid peroxidation, intracellular ROS accumulation, and decreased cell viability. Ferroptosis inhibitors rescued cell viability, confirming their role in cytotoxicity. These results implicate the HSA-Cys34 redox state is a promising avenue for precision medicine in DTC, shedding light on the prognostic relevance and therapeutic potential of HMA-induced ferroptosis. They emphasize the opportunity for personalized treatment strategies to advance the management of patients with DTC.

Detection of human serum albumin using a rare-earth nanosheet fluorescent probe based on intensity and lifetime signals

Human serum albumin (HSA) is an important biomarker for early disease diagnosis. Therefore, the detection of HSA in biological samples is important. In this study, for the sensitive detection of HSA, a fluorescent probe based on Eu(III)-doped yttrium hydroxide nanosheets was designed and sensitized by α-thiophenformyl acetone trifluoride as an antenna. The morphology and structure of the as-prepared nanosheet fluorescent probe were studied by transmission electron microscopy and atomic force microscopy. A detailed investigation of the fluorescence properties of the as-obtained nanosheet probe revealed that the Eu(III) emission intensity was linearly and selectively enhanced by the consecutive addition of HSA. Furthermore, the lifetime signal of this probe was enhanced with increasing concentration. The sensitivity of the nanosheet probe to HSA is discussed based on the results of ultraviolet–visible, fluorescence, and infrared spectral analyses, the findings demonstrate that the nanosheet fluorescent probe we prepared is a highly sensitive and selective tool for detecting HSA concentration with a high intensity and a large lifetime change.

Quetiapine Albumin Nanoparticles as an Efficacious Platform for Brain Deposition and Potentially Improved Antipsychotic Activity.

Quetiapine (QP) is a second-generation short-acting antipsychotic drug extensively metabolized in the liver, producing pharmacologically inactive metabolites and leading to diminished bioavailability. Therefore, this study aimed to develop an intravenous QP albumin nanoparticles (NPs) system for improving QP antipsychotic activity and brain targeting. QP-loaded albumin NPs were prepared by the desolvation method. The fabricated NPs were characterized in terms of particle size, zeta potential, entrapment efficiency (EE%), and in vitro drug release. In vivo pharmacokinetics and biodistribution in rats were studied. In addition, the antipsychotic activity of the optimized platform was also investigated. Human serum albumin (HSA) concentration, pH, and stirring time were modulated to optimize QP albumin NPs with a particle size of 103.54 ± 2.36 nm and a QP EE% of 96.32 ± 3.98%. In addition, the intravenous administration of QP albumin NPs facilitated QP brain targeting with a 4.9-fold increase in targeting efficiency compared to the oral QP solution. The QP albumin NPs improved the QP antipsychotic activity, indicated by suppressing rats' hypermobility and reducing the QP's extrapyramidal side effects. The obtained results proposed that intravenous QP- NPs could improve QP brain targeting and its antipsychotic efficiency.

Hydrolytic Metabolism of Withangulatin A Mediated by Serum Albumin Instead of Common Esterases in Plasma.

The oral bioavailability of withangulatin A (WA) is low and may undergo first-pass metabolism because of the presence of two esters bonds. This study aimed to identify the hydrolysis behavior and mechanism of WA, thus enriching its structure-pharmacokinetic relationship. The in vivo pharmacokinetic studies of WA in rats were first investigated, followed by in vitro assays including metabolic stability, phenotyping identification and metabolic kinetics assays. After screening out the responsible enzymes with higher catalytic capacity, molecular docking study was performed to demonstrate the interaction mode between WA and metabolic enzymes. Then, metabolites in human serum albumin (HSA) were identified by LC-TOF-MS/MS. In rats, the oral bioavailability of WA was only 2.83%. In vitro, WA was hydrolyzed in both rat and human plasma and could not be inhibited by selective esterase inhibitors. Physiologic concentration of HSA not recombinant human carboxylesterases (rhCES) could significantly hydrolyze WA, and it had a similar hydrolytic capacity with human plasma to WA. Furthermore, WA could stably bind to HSA by forming hydrogen bonds with Lys199 and Arg410, accompanied by the metabolic reaction of the lactone ring opening. The study showed that WA underwent obvious hydrolysis in rat and human plasma, which implied a strong first-pass effect. Serum albumin rather than common esterases primarily contributed to the hydrolytic metabolism of WA in plasma.

Quantitative determination of albumin and immunoglobulin in human serum using gold nanoclusters

In this experimental study, we developed a simple and selective approach to determine the concentrations of human serum albumin (HSA) and total amount of immunoglobulins (Ig) in real human serum (HS) sample using luminescent gold nanoclusters (Au NCs). In doing so, Au NCs were grown directly on the HS proteins without any sample pretreatment. We synthesized Au NCs on HSA and Ig and studied their photophysical properties. Using combined fluorescent and colorimetric assay we were able to obtain protein concentrations with a high degree of accuracy relative to techniques currently used in clinical diagnostics. We used method of standard additions to determine both HSA and Ig concentrations in HS by the Au NCs absorbance and fluorescence signals. A simple and cost-effective method developed in this work represents an excellent alternative to the techniques currently used in clinical diagnostics.

Hemopexin and albumin inhibit heme-induced macrophage activation while also enabling heme-LPS synergistic promotion of TNF production

Free heme released from hemoglobin contributes to exacerbated inflammation and tissue damage in hemolytic diseases. While a moderate level of free heme does not cause intravascular inflammation by itself, its presence during infection greatly enhances inflammation. Although specific serum proteins have been found to affect heme-induced inflammation, the selective contribution of serum proteins inhibiting macrophage activation by heme or, conversely, amplifying the production of cytokines by macrophages stimulated with heme and microbial molecules, is poorly defined. Here we identified a serum fraction containing proteins with >50 KDa which was capable of inhibiting heme-stimulated TNF production and capable of enabling TNF production under conditions of a heme-LPS synergy. The inhibition of heme-induced TNF production was mimicked by Hemopexin (Hx), human serum albumin (HSA), serum from Hx-knockout mice, and less efficiently by serum from albumin-knockout mice, but not by serum LDL. Hx and HSA inhibited heme-induced ROS generation, MAPK/ Syk phosphorylation and cell death. However, Hx and HSA each also promoted the synergistic relationship between heme and LPS upon TNF production. Serum from Hx-knockout mice was fully capable of enabling this synergy, while serum from albumin-knockout mice was less efficient to promote TNF production under these conditions. Low concentrations of HSA mimicked the ability of serum to enable heme-stimulated IL-1β production after LPS priming, while high concentrations inhibited it. Together, our findings indicate how heme inflammatory effects are restrained in the blood upon sterile hemolysis, yet exacerbate inflammation in the presence of microbes. Moreover, it is interesting to note that opposing effects of serum proteins on heme-induced macrophage activation were selected through evolution, with both effects exerted by Hx and albumin.

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.