Improved L-Asparaginase Properties and Reusability by Immobilization onto Functionalized Carbon Xerogels.

Enzyme immobilization can offer a range of significant advantages, including reusability, and increased selectivity, stability, and activity. In this work, a central composite design (CCD) of experiments and response surface methodology (RSM) were used to study, for the first time, the L-asparaginase (ASNase) immobilization onto functionalized carbon xerogels (CXs). The best results were achieved using CXs obtained by hydrothermal oxidation with nitric acid and subsequent heat treatment in a nitrogen flow at 600 °C (CX-OX-600). Under the optimal conditions (81 min of contact time, pH 6.2 and 0.36 g/L of ASNase), an immobilization yield (IY) of 100 % and relative recovered activity (RRA) of 103 % were achieved. The kinetic parameters obtained also indicate a 1.25-fold increase in the affinity of ASNase towards the substrate after immobilization. Moreover, the immobilized enzyme retained 97 % of its initial activity after 6 consecutive reaction cycles. All these outcomes confirm the promising properties of functionalized CXs as support for ASNase, bringing new insights into the development of an efficient and stable immobilization platform for use in the pharmaceutical industry, food industry, and biosensors.

Corrigendum: Ionic-liquid-based approaches to improve biopharmaceuticals downstream processing and formulation.

[This corrects the article DOI: 10.3389/fbioe.2023.1037436.].

Ionic-liquid-based approaches to improve biopharmaceuticals downstream processing and formulation.

The emergence of biopharmaceuticals, including proteins, nucleic acids, peptides, and vaccines, revolutionized the medical field, contributing to significant advances in the prophylaxis and treatment of chronic and life-threatening diseases. However, biopharmaceuticals manufacturing involves a set of complex upstream and downstream processes, which considerably impact their cost. In particular, despite the efforts made in the last decades to improve the existing technologies, downstream processing still accounts for more than 80% of the total biopharmaceutical production cost. On the other hand, the formulation of biological products must ensure they maintain their therapeutic performance and long-term stability, while preserving their physical and chemical structure. Ionic-liquid (IL)-based approaches arose as a promise alternative, showing the potential to be used in downstream processing to provide increased purity and recovery yield, as well as excipients for the development of stable biopharmaceutical formulations. This manuscript reviews the most important progress achieved in both fields. The work developed is critically discussed and complemented with a SWOT analysis.

New autonomous and self-signaling biosensing device for sarcosine detection.

An early diagnosis is the gold standard for cancer survival. Biosensors have proven their effectiveness in monitoring cancer biomarkers but are still limited to a series of requirements. This work proposes an integrated power solution, with an autonomous and self-signaling biosensing device. The biorecognition element is produced in situ by molecular imprinting to detect sarcosine, a known biomarker for prostate cancer. The biosensor was assembled on the counter-electrode of a dye-sensitized solar cell (DSSC), simultaneously using EDOT and Pyrrole as monomers for the biomimetic process and the catalytic reduction of triiodide in the DSSC. After the rebinding assays, the hybrid DSSC/biosensor displayed a linear behavior when plotting the power conversion efficiency (PCE) and the charge transfer resistance (RCT) against the logarithm of the concentration of sarcosine. The latter obtained a sensitivity of 0.468 Ω/decade of sarcosine concentration, with a linear range between 1 ng/mL and 10 µg/mL, and a limit of detection of 0.32 ng/mL. When interfacing an electrochromic cell, consisting of a PEDOT-based material, with the hybrid device, a color gradient between 1 ng/mL and 10 µg/mL of sarcosine was observed. Thus, the device can be used anywhere with access to a light source, completely equipment-free, suitable for point-of-care analysis and capable of detecting sarcosine within a range of clinical interest.

Engineering graphene-based electrodes for optical neural stimulation.

Graphene-based materials (GBMs) have been investigated in recent years with the aim of developing flexible interfaces to address a range of neurological disorders, where electrical stimulation may improve brain function and tissue regeneration. The recent discovery that GBM electrodes can generate an electrical response upon light exposure has inspired the development of non-genetic approaches capable of selectively modulating brain cells without genetic manipulation (i.e., optogenetics). Here, we propose the conjugation of graphene with upconversion nanoparticles (UCNPs), which enable wireless transcranial activation using tissue-penetrating near-infrared (NIR) radiation. Following a design of experiments approach, we first investigated the influence of different host matrices and dopants commonly used to synthesize UCNPs in the electrical response of graphene. Two UCNP formulations achieving optimal enhancement of electrical conductivity upon NIR activation at λ = 780 or 980 nm were identified. These formulations were then covalently attached to graphene nanoplatelets following selective hydroxyl derivatization. The resulting nanocomposites were evaluated in vitro using SH-SY5Y human neuroblastoma cells. NIR activation at λ = 980 nm promoted cell proliferation and downregulated neuronal and glial differentiation markers, suggesting the potential application of GBMs in minimally invasive stimulation of cells for tissue regeneration.

New liquid supports in the development of integrated platforms for the reuse of oxidative enzymes and polydopamine production.

Polydopamine (PDA), a bioinspired polymer from mussel adhesive proteins, has attracted impressive attention as a novel coating for (nano) materials with an adequate conformal layer and adjustable thickness. Currently, PDA is obtained from dopamine chemical oxidation under alkaline conditions, limiting its use in materials sensible to alkaline environments. Envisaging a widespread use of PDA, the polymerization of dopamine by enzymatic catalysis allows the dopamine polymerization in a large range of pHs, overcoming thus the limitations of conventional chemical oxidation. Moreover, the conventional method of polymerization is a time-consuming process and produces PDA films with poor stability, which restricts its applications. On the other hand, the main bottleneck of enzyme-based biocatalytic processes is the high cost of the single use of the enzyme. In this work, laccase was used to catalyse dopamine polymerization. To improve its performance, a liquid support for integrating the laccase and its reuse together with the PDA production and recovery was developed using aqueous biphasic systems (ABS). Firstly, dopamine polymerization by laccase was optimized in terms of pH, temperature and initial dopamine concentration. It was demonstrated that the highest enzymatic polymerization of dopamine was achieved at pH 5.5, 30°C and 2 mg ml-1 of dopamine. Then, ABS composed of polymers, salts and ionic liquids were evaluated to optimize the laccase confinement in one phase while PDA is recovered in the opposite phase. The most promising ABS allowing the separation of laccase from the reaction product is composed of polypropylene glycol (400 g mol-1) and K2HPO4. The polymerization of dopamine in ABS leads to a remarkable improvement of polymerization of 3.9-fold in comparison to the conventional chemical PDA polymerization. The phase containing the confined laccase was reused for four consecutive reaction cycles, with a relative polymerization of 68.9% in the last cycle. The results of this work proved that ABS are a promising approach to create a liquid support for enzyme reuse allowing the process intensification efforts. The use of biocatalysts in ABS emerges as sustainable and alternative platforms from environmental and techno-economic points of view.

An Electrochemical Immunosensor Based on Carboxylated Graphene/SPCE for IgG-SARS-CoV-2 Nucleocapsid Determination.

The COVID-19 pandemic has emphasized the importance and urgent need for rapid and accurate diagnostic tests for detecting and screening this infection. Our proposal was to develop a biosensor based on an ELISA immunoassay for monitoring antibodies against SARS-CoV-2 in human serum samples. The nucleocapsid protein (N protein) from SARS-CoV-2 was employed as a specific receptor for the detection of SARS-CoV-2 nucleocapsid immunoglobulin G. N protein was immobilized on the surface of a screen-printed carbon electrode (SPCE) modified with carboxylated graphene (CG). The percentage of IgG-SARS-CoV-2 nucleocapsid present was quantified using a secondary antibody labeled with horseradish peroxidase (HRP) (anti-IgG-HRP) catalyzed using 3,3',5,5'-tetramethylbenzidine (TMB) mediator by chronoamperometry. A linear response was obtained in the range of 1:1000-1:200 v/v in phosphate buffer solution (PBS), and the detection limit calculated was 1:4947 v/v. The chronoamperometric method showed electrical signals directly proportional to antibody concentrations due to antigen-antibody (Ag-Ab) specific and stable binding reaction.

Aliivibrio fischeri L-Asparaginase production by engineered Bacillus subtilis: a potential new biopharmaceutical.

L-Asparaginase (L-ASNase) is an enzyme applied in the treatment of lymphoid malignancies. However, an innovative L-ASNase with high yield and lower side effects than the commercially available preparations are still a market requirement. Here, a new-engineered Bacillus subtilis strain was evaluated for Aliivibrio fischeri L-ASNase II production, being the bioprocess development and the enzyme characterization studied. The pBS0E plasmid replicative in Bacillus sp and containing PxylA promoter inducible by xylose and its repressive molecule sequence (XylR) was used for the genetic modification. Initially, cultivations were carried out in orbital shaker, and then the process was scaled up to stirred tank bioreactor (STB). After the bioprocess, the cells were recovered and submitted to ultrasound sonication for cells disruption and intracellular enzyme recovery. The enzymatic extract was characterized to assess its biochemical, kinetic and thermal properties using L-Asparagine and L-Glutamine as substrates. The results indicated the potential enzyme production in STB achieving L-ASNase activity up to 1.539 U mL-1. The enzymatic extract showed an optimum pH of 7.5, high L-Asparagine affinity (Km = 1.2275 mmol L-1) and low L-Glutaminase activity (0.568-0.738 U mL-1). In addition, thermal inactivation was analyzed by two different Kinect models to elucidate inactivation mechanisms, low kinetic thermal inactivation constants for 25 ºC and 37 ºC (0.128 and 0.148 h-1, respectively) indicate an elevated stability. The findings herein show that the produced recombinant L-ASNase has potential to be applied for pharmaceutical purposes.

Immobilization and Characterization of L-Asparaginase over Carbon Xerogels.

L-asparaginase (ASNase) is an aminohydrolase currently used in the pharmaceutical and food industries. Enzyme immobilization is an exciting option for both applications, allowing for a more straightforward recovery and increased stability. High surface area and customizable porosity make carbon xerogels (CXs) promising materials for ASNase immobilization. This work describes the influence of contact time, pH, and ASNase concentration on the immobilization yield (IY) and relative recovered activity (RRA) using the Central Composite Design methodology. The most promising results were obtained using CX with an average pore size of 4 nm (CX-4), reaching IY and RRA of 100%. At the optimal conditions (contact time 49 min, pH 6.73, and [ASNase] 0.26 mg·mL-1), the ASNase-CXs biocomposite was characterized and evaluated in terms of kinetic properties and operational, thermal, and pH stabilities. The immobilized ASNase onto CX-4 retained 71% of its original activity after six continuous reaction cycles, showed good thermal stability at 37 °C (RRA of 91% after 90 min), and was able to adapt to both acidic and alkaline environments. Finally, the results indicated a 3.9-fold increase in the immobilized ASNase affinity for the substrate, confirming the potential of CXs as a support for ASNase and as a cost-effective tool for subsequent use in the therapeutic and food sectors.

Enhanced Enzyme Reuse through the Bioconjugation of L-Asparaginase and Silica-Based Supported Ionic Liquid-like Phase Materials.

L-asparaginase (ASNase) is an amidohydrolase that can be used as a biopharmaceutical, as an agent for acrylamide reduction, and as an active molecule for L-asparagine detection. However, its free form displays some limitations, such as the enzyme's single use and low stability. Hence, immobilization is one of the most effective tools for enzyme recovery and reuse. Silica is a promising material due to its low-cost, biological compatibility, and tunable physicochemical characteristics if properly functionalized. Ionic liquids (ILs) are designer compounds that allow the tailoring of their physicochemical properties for a given task. If properly designed, bioconjugates combine the features of the selected ILs with those of the support used, enabling the simple recovery and reuse of the enzyme. In this work, silica-based supported ionic liquid-like phase (SSILLP) materials with quaternary ammoniums and chloride as the counterion were studied as novel supports for ASNase immobilization since it has been reported that ammonium ILs have beneficial effects on enzyme stability. SSILLP materials were characterized by elemental analysis and zeta potential. The immobilization process was studied and the pH effect, enzyme/support ratio, and contact time were optimized regarding the ASNase enzymatic activity. ASNase-SSILLP bioconjugates were characterized by ATR-FTIR. The bioconjugates displayed promising potential since [Si][N3444]Cl, [Si][N3666]Cl, and [Si][N3888]Cl recovered more than 92% of the initial ASNase activity under the optimized immobilization conditions (pH 8, 6 × 10-3 mg of ASNase per mg of SSILLP material, and 60 min). The ASNase-SSILLP bioconjugates showed more enhanced enzyme reuse than reported for other materials and immobilization methods, allowing five cycles of reaction while keeping more than 75% of the initial immobilized ASNase activity. According to molecular docking studies, the main interactions established between ASNase and SSILLP materials correspond to hydrophobic interactions. Overall, it is here demonstrated that SSILLP materials are efficient supports for ASNase, paving the way for their use in the pharmaceutical and food industries.

Molecularly Imprinted Solid Phase Extraction Aiding the Analysis of Disease Biomarkers.

Low concentrations of biomarkers as well as the complexity of biological samples make the clinical diagnoses of several diseases a challenging task. Sample preparation protocols remain a fundamental piece in the puzzle of analytical processes, and smart sorbents including molecularly imprinted polymers (MIPs) have been successfully used in this case. In this review, we depict the state of art for the rational design of MIPs to be used in solid phase extraction of disease biomarkers from biological samples. The topics are divided into (1) strategies for MIP syntheses, (2) setups for sample preparation protocols with MIPs, (3) the applications of these combined principles in the analyses of different classes of disease biomarkers, and (4) remaining challenges and future trends for the application of Molecular Imprinting Technology in sample preparation for clinical diagnosis.

Paper-Based Biosensors for COVID-19: A Review of Innovative Tools for Controlling the Pandemic.

The appearance and quick spread of the new severe acute respiratory syndrome coronavirus disease, COVID-19, brought major societal challenges. Importantly, suitable medical diagnosis procedures and smooth clinical management of the disease are an emergent need, which must be anchored on novel diagnostic methods and devices. Novel molecular diagnostic tools relying on nucleic acid amplification testing have emerged globally and are the current gold standard in COVID-19 diagnosis. However, the need for widespread testing methodologies for fast, effective testing in multiple epidemiological scenarios remains a crucial step in the fight against the COVID-19 pandemic. Biosensors have previously shown the potential for cost-effective and accessible diagnostics, finding applications in settings where conventional, laboratorial techniques may not be readily employed. Paper- and cellulose-based biosensors can be particularly relevant in pandemic times, for the renewability, possibility of mass production with sustainable methodologies, and safe environmental disposal. In this review, paper-based devices and platforms targeting SARS-CoV-2 are showcased and discussed, as a means to achieve quick and low-cost PoC diagnosis, including detection methodologies for viral genomic material, viral antigen detection, and serological antibody testing. Devices targeting inflammatory markers relevant for COVID-19 are also discussed, as fast, reliable bedside diagnostic tools for patient treatment and follow-up.

Superior operational stability of immobilized L-asparaginase over surface-modified carbon nanotubes.

L-asparaginase (ASNase, EC 3.5.1.1) is an enzyme that catalyzes the L-asparagine hydrolysis into L-aspartic acid and ammonia, being mainly applied in pharmaceutical and food industries. However, some disadvantages are associated with its free form, such as the ASNase short half-life, which may be overcome by enzyme immobilization. In this work, the immobilization of ASNase by adsorption over pristine and modified multi-walled carbon nanotubes (MWCNTs) was investigated, the latter corresponding to functionalized MWCNTs through a hydrothermal oxidation treatment. Different operating conditions, including pH, contact time and ASNase/MWCNT mass ratio, as well as the operational stability of the immobilized ASNase, were evaluated. For comparison purposes, data regarding the ASNase immobilization with pristine MWCNT was detailed. The characterization of the ASNase-MWCNT bioconjugate was addressed using different techniques, namely Transmission Electron Microscopy (TEM), Thermogravimetric Analysis (TGA) and Raman spectroscopy. Functionalized MWCNTs showed promising results, with an immobilization yield and a relative recovered activity of commercial ASNase above 95% under the optimized adsorption conditions (pH 8, 60 min of contact and 1.5 × 10-3 g mL-1 of ASNase). The ASNase-MWCNT bioconjugate also showed improved enzyme operational stability (6 consecutive reaction cycles without activity loss), paving the way for its use in industrial processes.

Advances Achieved by Ionic-Liquid-Based Materials as Alternative Supports and Purification Platforms for Proteins and Enzymes.

Ionic liquids (ILs) have been applied in several fields in which enzymes and proteins play a noteworthy role, for instance in biorefinery, biotechnology, and pharmaceutical sciences, among others. Despite their use as solvents and co-solvents, their combination with materials for protein- and enzyme-based applications has raised significant attention in the past few years. Among them, significant advances were brought by supported ionic liquids (SILs), in which ILs are introduced to modify the surface and properties of materials, e.g., as ligands when covalently bond or when physiosorbed. SILs have been mainly investigated as alternative supports for enzymes in biocatalysis and as new supports in preparative liquid chromatography for the purification of high-value proteins and enzymes. In this manuscript, we provide an overview on the most relevant advances by using SILs as supports for enzymes and as purification platforms for a variety of proteins and enzymes. The interaction mechanisms occurring between proteins and SILs/ILs are highlighted, allowing the design of efficient processes involving SILs. The work developed is discussed in light of the respective development phase and innovation level of the applied technologies. Advantages and disadvantages are identified, as well as the missing links to pave their use in relevant applications.

Biosensors for European Zoonotic Agents: A Current Portuguese Perspective.

Emerging and recurrent outbreaks caused by zoonotic agents pose a public health risk. They result in morbidity and mortality in humans and significant losses in the livestock and food industries. This highlights the need for rapid surveillance methods. Despite the high reliability of conventional pathogen detection methods, they have high detection limits and are time-consuming and not suitable for on-site analysis. Furthermore, the unpredictable spread of zoonotic infections due to a complex combination of risk factors urges the development of innovative technologies to overcome current limitations in early warning and detection. Biosensing, in particular, is highlighted here, as it offers rapid and cost-effective devices for use at the site of infection while increasing the sensitivity of detection. Portuguese research in biosensors for zoonotic pathogens is the focus of this review. This branch of research produces exciting and innovative devices for the study of the most widespread pathogenic bacteria. The studies presented here relate to the different classes of pathogens whose characteristics and routes of infection are also described. Many advances have been made in recent years, and Portuguese research teams have increased publications in this field. However, biosensing still needs to be extended to other pathogens, including potentially pandemic viruses. In addition, the use of biosensors as part of routine diagnostics in hospitals for humans, in animal infections for veterinary medicine, and food control has not yet been achieved. Therefore, a convergence of Portuguese efforts with global studies on biosensors to control emerging zoonotic diseases is foreseen for the future.

L-asparaginase production review: bioprocess design and biochemical characteristics.

In the past decades, the production of biopharmaceuticals has gained high interest due to its great sensitivity, specificity, and lower risk of negative effects to patients. Biopharmaceuticals are mostly therapeutic recombinant proteins produced through biotechnological processes. In this context, L-asparaginase (L-asparagine amidohydrolase, L-ASNase (E.C. 3.5.1.1)) is a therapeutic enzyme that has been abundantly studied by researchers due to its antineoplastic properties. As a biopharmaceutical, L-ASNase has been used in the treatment of acute lymphoblastic leukemia (ALL), acute myeloblastic leukemia (AML), and other lymphoid malignancies, in combination with other drugs. Besides its application as a biopharmaceutical, this enzyme is widely used in food processing industries as an acrylamide mitigation agent and as a biosensor for the detection of L-asparagine in physiological fluids at nano-levels. The great demand for L-ASNase is supplied by recombinant enzymes from Escherichia coli and Erwinia chrysanthemi. However, production processes are associated to low yields and proteins associated to immunogenicity problems, which leads to the search for a better enzyme source. Considering the L-ASNase pharmacological and food importance, this review provides an overview of the current biotechnological developments in L-ASNase production and biochemical characterization aiming to improve the knowledge about its production. KEY POINTS: • Microbial enzyme applications as biopharmaceutical and in food industry • Biosynthesis process: from the microorganism to bioreactor technology • Enzyme activity and kinetic properties: crucial for the final application.

Sustainable liquid supports for laccase immobilization and reuse: Degradation of dyes in aqueous biphasic systems.

Novel liquid supports for enzyme immobilization and reuse based on aqueous biphasic systems (ABS) constituted by cholinium-based ionic liquids (ILs) and polymers for the degradation of dyes are here proposed. The biocatalytic reaction for dye decolorization using laccase occured in the biphasic medium, with the enzyme being "supported" in the IL-rich phase and the dye and degradation products being enriched in the polymer-rich phase. An initial screening of the laccase activity in aqueous solutions of ABS constituents, namely cholinium dihydrogen citrate ([Ch][DHC]), cholinium dihydrogen phosphate ([Ch][DHP]), cholinium acetate ([Ch][Acet]), polypropylene glycol 400 (PPG 400), polyethylene glycol 400 (PEG 400) and K2 HPO4 was carried out. Compared to the buffered control, a relative laccase activity of up to 170%, 257%, and 530% was observed with PEG 400, [Ch][DHP], and [Ch][DHC], respectively. These ABS constituents were then investigated for the in situ enzymatic biodegradation of the Remazol Brilliant Blue R (RBBR) dye. At the optimized conditions, the ABS constituted by PPG 400 at 46 wt% and [Ch][DHC] at 16 wt% leads to the complete degradation of the RBBR dye, further maintaining the enzyme activity. This ABS also allows an easy immobilization, recovery, and reuse of the biocatalyst for six consecutive reaction cycles, achieving a degradation yield of the dye of 96% in the last cycle. In summary, if properly designed, high enzymatic activities and reaction yields are obtained with ABS as liquid supports, while simultaneously overcoming the safety and environmental concerns of conventional organic solvents used in liquid-liquid heterogeneous reactions, thus representing more sustainable biocatalytic processes.

A passive direct methanol fuel cell as transducer of an electrochemical sensor, applied to the detection of carcinoembryonic antigen.

This work describes an electrochemical sensor with a biomimetic plastic antibody film for carcinoembryonic antigen (CEA, an important biomarker in colorectal cancer), integrated in the electrical circuit of a direct methanol fuel cell (DMFC), working in passive mode and used herein as power supply and signal transducer. In detail, the sensing layer for CEA consisted of a Fluorine-doped Tin Oxide (FTO) conductive glass substrate - connected to the negative pole side of the DMFC - with a conductive poly (3,4-ethylenedioxythiophene) (PEDOT) layer and a polypyrrol (PPy) molecularly-imprinted polymer (MIP), assembled in-situ. This sensing element is then closed using a cover FTO-glass, hold in place with a clip, connected to the positive side of the DMFC. When compared with control DMFCs, the power curves of DMFC/Sensor integrated system showed decreased power values due to the MIP layer interfaced in the electrical circuit, also displaying high stability signals. The DMFC/Sensor was further calibrated at room temperature, in different medium (buffer, a synthetic physiological fluid model and Cormay® serum), showing linear responses over a wide concentration range, with a limit of detection of 0.08 ng/mL. The DMFC/Sensor presented sensitive data, with linear responses from 0.1 ng/mL to 100 µg/mL and operating well in the presence of human serum. Overall, the results obtained evidenced the possibility of using a DMFC as a transducing element in an electrochemical sensor, confirming the sensitive and selective readings of the bio (sensing) imprinted film. This integration paves the way towards fully autonomous electrochemical devices, in which the integration of the sensor inside the fuel cell may be a subsequent direction.

Recent Strategies and Applications for l-Asparaginase Confinement.

l-asparaginase (ASNase, EC 3.5.1.1) is an aminohydrolase enzyme with important uses in the therapeutic/pharmaceutical and food industries. Its main applications are as an anticancer drug, mostly for acute lymphoblastic leukaemia (ALL) treatment, and in acrylamide reduction when starch-rich foods are cooked at temperatures above 100 °C. Its use as a biosensor for asparagine in both industries has also been reported. However, there are certain challenges associated with ASNase applications. Depending on the ASNase source, the major challenges of its pharmaceutical application are the hypersensitivity reactions that it causes in ALL patients and its short half-life and fast plasma clearance in the blood system by native proteases. In addition, ASNase is generally unstable and it is a thermolabile enzyme, which also hinders its application in the food sector. These drawbacks have been overcome by the ASNase confinement in different (nano)materials through distinct techniques, such as physical adsorption, covalent attachment and entrapment. Overall, this review describes the most recent strategies reported for ASNase confinement in numerous (nano)materials, highlighting its improved properties, especially specificity, half-life enhancement and thermal and operational stability improvement, allowing its reuse, increased proteolysis resistance and immunogenicity elimination. The most recent applications of confined ASNase in nanomaterials are reviewed for the first time, simultaneously providing prospects in the described fields of application.

Recovery of immunoglobulin G from rabbit serum using κ-carrageenan-modified hybrid magnetic nanoparticles.

Immunoglobulin G (IgG) has been used in the treatment of cancer, autoimmune diseases and neurological disorders, however, the current technologies to purify and recover IgG from biological media are of high-cost and time-consuming, resulting in high-cost products. In this sense, the search for cost-effective technologies to obtain highly pure and active IgG is highly required. The present work proposes a simple and efficient method for the purification and recovery of IgG from rabbit serum using magnetic iron oxide nanoparticles (magnetite, Fe3O4) coated with hybrid shells of a siliceous material modified with the anionic polysaccharide κ-carrageenan. Experimental parameters such as pH, contact time between the hybrid magnetic nanoparticles (HMNPs) and rabbit serum, and total protein concentration or dilution factor of serum were evaluated. The best results were achieved at pH 5.0, with a contact time of 60 min and using a rabbit serum with a total protein concentration of 4.8 mg·mL-1. Under these conditions, it was obtained an IgG purification factor and adsorption yield onto the HMNPs of 3.0 and 90%, respectively. The desorption of IgG from the HMNPs was evaluated using two strategies: a KCl aqueous solution and buffered aqueous solutions. Comparing to the initial rabbit serum, an IgG purification factor of 2.7 with a recovery yield of 74% were obtained using a buffered aqueous solution at pH 7.0. After desorption, the secondary structure of IgG and other proteins was evaluated by circular dichroism and no changes in the secondary structure were observed, meaning that the IgG integrity is kept after the adsorption and desorption steps. In summary, the application of HMNPs in the purification of IgG from serum samples has a high potential as a new downstream platform.