Nanomaterials and Fluorescent Probes: Innovations in Bovine Albumin Detection

Proteins play diverse roles in biology, necessitating their precise recognition for biological studies and clinical diagnosis. Accurate detection of proteins is crucial, demanding highly sensitive methods. Albumin, often used to quantify constituents in living systems, requires precise determination. Existing methods like Biuret, Lowry, Bradford, and Bromocresol Green suffer from limitations such as dynamic range, sensitivity, and detection limits. Overcoming these constraints, new methods are being developed.

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Fig. 1 The structure of HSA (Jahanban-Esfahlan A., et al. 2019).

Serum albumins, constituting 52–62% of total water-soluble fraction proteins in blood plasma, are vital bio-macromolecules with circulatory, nutritional, and physiological functions. These proteins, predominantly human serum albumin (HSA) and bovine serum albumin (BSA), act as transporters, distributing various substances in the body. HSA, abundant in the human circulatory system, is a heart-shaped globular protein with a molecular weight of 66.8 kDa. It comprises 586 amino acids folding into a three-dimensional structure with three domains (I, II, and III), each containing subdomains A and B. HSA's main binding sites, Sulow's sites I and II, are located in subdomains IIA and IIIA. HSA plays a crucial role in binding, transporting, and delivering steroids, fatty acids, bilirubin, and porphyrins. Notably, albumin-based nanoparticles, exemplified by Abraxane, have been developed for efficient drug delivery.

Similarly, BSA, structurally akin to HSA, is often used as a model protein. Comprising 583 amino acids stabilized by 17 disulfide bridges, BSA shares a heart-shaped structure with three domains I-III, each featuring subdomains A and B. In subdomains IB and IIA, BSA contains two Tryptophan (Trp) amino acid residues (Trp-134 and Trp-212). BSA's resemblance to HSA makes it a valuable substitute in research. Both albumins play essential roles in biomedical applications and serve as foundational components for innovative drug delivery systems.

Determination of BSA: Advances in Quantification Techniques

BSA plays a crucial role in medical applications and enzymatic reactions, serving as a protein reagent to measure protein concentration and as a standard protein in immunochemical tests. Additionally, BSA finds use as a dietary protein. The demand for accurate, fast, sensitive, and reproducible protein analysis methods has driven research into advanced techniques.

Capillary Electrophoresis for BSA Quantification

Decades ago, capillary electrophoresis (CE) emerged as a rapid and sensitive method for BSA quantification. Laser-induced fluorescence (LIF) schemes, particularly those utilizing aromatic amino acids in proteins, demonstrated substantial improvements in detection sensitivity. With a wavelength of 275.4 nm, the CE-LIF detecting platform achieved a limit of detection (LOD) of 1 × 10^⁻10 M for conalbumin.

Optimization studies by researchers aimed to enhance the reproducibility, accuracy, and speed of CE methods for BSA assays. Parameters like buffer concentration, pH, capillary dimensions, and additives were considered, leading to optimal detection conditions. This method showed a validation coefficient of variation below 7.59% for BSA concentrations ranging from 25 to 1000 μg/ml.

Capillary Zone Electrophoresis (CZE)

Traditional gel electrophoresis (GE) is known for being labor-intensive, time-consuming, and unsuitable for quantifying diverse protein samples. In contrast, capillary zone electrophoresis (CZE) addresses these drawbacks, offering rapid separation and analysis of charged molecules.

BSA Recognition through Fluorescent Materials

Fluorescent Probes

Fluorescence spectroscopy has emerged as a powerful technique for studying biomolecular interactions. Various fluorescent probes have been developed, such as diketopyrrolopyrrole derivatives, chemically converted graphene (CCG)-dye complexes, and dye-metal complexes.

Diketopyrrolopyrrole (DPP) and Its Derivatives

DPP derivatives, particularly 1,4-diketo-3,6-diphenylpyrrolo[3,4-c]pyrrole (DPP), exhibit strong fluorescence and have been extensively employed in various applications. A novel NIR emissive fluorophore, DPPAM, demonstrated a 'turn-on' response for BSA, showcasing high sensitivity and NIR emission ranging from 600 to 850 nm. DPP derivatives functionalized with electron-donating groups have shown AIE activity, making them valuable in biological applications.

In another study, synthesized DPP-based compounds demonstrated AIE properties and pH-sensitive fluorescence. DPP1, with diethylamino groups, exhibited a specific AIE property under alkaline conditions, acting as a pH sensor. DPP2, an ammonium compound, showed no AIE activity in solution but demonstrated significant fluorescence enhancement in the presence of BSA in a solid state.

Chemically Converted Graphene (CCG)-Dye Complexes

CCG-dye complexes have gained attention for their fluorescence properties. Cyanin 3 (Cy3) dye, when absorbed on CCG sheets, exhibited efficient quenching. However, in the presence of BSA, fluorescence intensity increased, making it a suitable candidate for discriminating BSA. Similarly, squaraine (SQ) dyes showed enhanced fluorescence intensity in the presence of BSA.

Dye-Metal Complexes

Dye-metal complexes, especially those involving transition metals, provide advantages in terms of sensitivity and selectivity. Ruthenium (II)-polypyridine complexes demonstrated luminescence sensor properties for BSA detection, relying on enhanced luminescence intensity and considerable hypsochromic shifts in the presence of albumin.

Other Fluorescent Probes

Various other fluorescent probes, such as 9,10-anthraquinone (AQ), Nile blue, 2,4-dihydroxyl-3-iodo salicylaldehyde azine (DISA), and enoxacin (ENX)-aluminum (Al³⁺) complex, have been explored for BSA detection. These probes offer advantages such as improved photochemical reactivity, sensitivity, and selectivity.

Fluorescence Resonance Energy Transfer (FRET) for BSA Detection

FRET has emerged as a powerful and sensitive spectroscopic technique for BSA detection. Utilizing rhodamine-6G (R6G) and gold nanoparticles (AuNPs) as donor and acceptor molecules, respectively, a FRET-based method demonstrated efficient BSA recognition. The addition of BSA induced aggregation of AuNPs, leading to fluorescence intensity recovery of R6G. This FRET-based method provided a rapid analysis with high selectivity in serum and urine samples.

Nanomaterial-Based Sensors

Nanomaterial-based sensors have gained significant attention, and among them, magnetic nanoparticles (MNPs) and gold-based nanomaterials, such as gold nanoparticles (AuNPs) and gold nanorods (Au NRs), play crucial roles in biological applications.

AuNPs, known for their biocompatibility and unique properties, have been utilized in the fabrication of various sensors. Surface-enhanced Raman scattering (SERS) substrates using AuNPs demonstrated stable signal intensity for over 45 days, allowing reliable measurement of biomolecules like albumin and creatinine. Modified gold electrodes coated with 2D silica network/citrate-capped AuNPs-poly diallyl dimethyl ammonium chloride (GE–2DSN-CAuNPs-PDDA) exhibited high sensitivity for ultra-trace determination of BSA.

Gold nanorods (Au NRs) with excellent radiative and nonradiative properties have been employed in label-free fluorescent biosensors for BSA and calf thymus DNA detection. The unique properties of Au NRs allow for the development of multifunctional nanoprobes with high specific surface area.

Zinc oxide (ZnO) nanomaterials, including microwires and nanospheres, have shown promise in biosensing. ZnO microwires integrated into a customized electronic platform demonstrated real-time detection of BSA binding, providing a proof of concept for biosensing applications. Solution-processed zinc oxide nanospheres exhibited ultra-low BSA detection with a sensitivity of 0.126 nA/pM and a fast response time of 5 seconds.

Graphene oxide (GO) and carbon nanotubes (CNTs) have also been investigated. GO exhibited π-π stacking interaction with the anticancer drug 10-hydroxy camptothecin (HCPT), enhancing the fluorescence response to BSA. Multiwalled carbon nanotubes (MWCNTs) have been utilized in molecularly imprinted membranes (MIMs) and other sensing devices for selective and sensitive BSA detection.

Quantum dots (QDs) showed promise in fluorescence-based sensors for BSA detection. CdTe QDs exhibited electrochemiluminescence (ECL) for BSA determination, offering a linear response in a low concentration range. Additionally, InGaP/ZnS core-shell QDs bioconjugated with anti-BSA antibodies served as fluorescent probes, demonstrating affinity for BSA detection through photoluminescence quenching.

In conclusion, detecting and quantifying bovine albumin, a key protein in cow-derived products, is crucial for food safety and biomedical assays. Modern nanotechnology-based methods, employing metallic or non-metallic nanoparticles like AuNPs and MNPs, offer high accuracy, sensitivity, and efficiency. Combining nanomaterials with spectroscopic techniques enables ultra-precise albumin detection. Despite notable advancements, current methods fall short for industrial applications. Further research and development are essential to create novel, practical albumin detection methods for diverse fields.

Reference

1. Jahanban-Esfahlan A., et al. Recent developments in the detection of bovine serum albumin. International Journal of Biological Macromolecules. 2019, 138: 602-617.