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1.
ACS Appl Bio Mater ; 5(1): 82-96, 2022 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-35014811

RESUMO

The lateral flow assay (LFA) is a point-of-care diagnostic test commonly available in an over-the-counter format because of its simplicity, speed, low cost, and portability. The reporter particles in these assays are among their most significant components because they perform the diagnostic readout and dictate the test's sensitivity. Today, gold nanoparticles are frequently used as reporters, but recent work focusing on photoluminescent-based reporter technologies has pushed LFAs to better performance. These efforts have focused specifically on reporters made of organic fluorophores, quantum dots, lanthanide chelates, persistent luminescent phosphors, and upconversion phosphors. In most cases, photoluminescent reporters show enhanced sensitivity compared to conventional gold nanoparticle-based assays. Here, we examine the advantages and disadvantages of these different reporters and highlight their potential benefits in LFAs. Our assessment shows that photoluminescent-based LFAs can not only reach lower detection limits than LFAs with traditional reporters, but they also can be capable of quantitative and multiplex analyte detection. As a result, the photoluminescent reporters make LFAs well-suited for medical diagnostics, the food and agricultural industry, and environmental testing.

2.
MAbs ; 13(1): 1980178, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34662534

RESUMO

Antibodies and Fc fusion proteins are a rapidly growing class of pharmaceuticals. Cell culture and purification process development and operation require frequent measurement of product concentrations, commonly by complex enzyme-linked immunosorbent assay and high-performance liquid chromatography methods. Here we report a fast (<30 s), and simple antibody Fc assay based on mix-and-read reporting by fluorescence emission. A soluble fluorescein-labeled Fc-affinity reporter produced by standard peptide synthesis is mixed with an Fc-containing sample to produce an immediate shift in both fluorescence polarization and intensity, compatible with on- and at-line measurements and microbioreactor monitoring. We observed significant shifts in fluorescence intensity in Chinese hamster ovary cell culture fluid spiked with IgG and detected an adalimumab biosimilar down to 100 ng/mL (10-4 g/L), despite the interferents in the complex sample matrix. Neither the fluorescence polarization nor the fluorescence intensity assay is significantly affected by the addition of clarified lysate of 2 million CHO-k1 cells/mL, suggesting applicability even to cultures of low viability. Biochemical and molecular docking approaches suggest that the fluorescence intensity enhancement is caused by changes in the fluorophore's local microenvironment upon binding to IgG Fc, especially by interactions with Fc His433.Abbreviations: CCF: Cell Culture Fluid; CHO: Chinese Hamster Ovary cells; ELISA: Enzyme Linked Immunosorbent Assay; Fc: Fragment Crystallizable of antibody; HPLC: High-Performance Liquid Chromatography; HPßCD: hydroxypropyl-ß-cyclodextrin; IgG: ImmunoglobulinG; mAb: Monoclonal Antibody; PBS: Phosphate-Buffered Saline; PDB: Protein Data Bank; SpA: Staphylococcal protein A; SpG: Staphylococcal protein G.

3.
Artigo em Inglês | MEDLINE | ID: mdl-34476713

RESUMO

Understanding the transport of sediments in urban estuaries and their effects on water quality and microorganisms is a convergent challenge that has yet to be addressed especially as a result of natural hazards that affect the hydrodynamics of estuarine systems. This study provides a holistic view of the longitudinal nature and character of sediment in an urban estuary, the Galveston Bay Estuary System (GBES), under daily and extreme flow regimes and presents the results of water and sediment sampling after Hurricane Harvey. The sediment sampling quantified total suspended sediment (TSS) concentrations, metal concentrations, and the diversity of microbial communities. The results revealed the impact of the substantial sediment loads that were transported into the GBES in terms of sediment grain type, the spatial distribution of trace metals, and the diversity of microbial communities. A measurable shift in the percentage of silt relative to historical norms was noted in the GBES after Hurricane Harvey. Not only did sediment metal data confirms this shift and its ensuing impact on metal concentrations; microbial data provided ample evidence of the effect of leaks and spills from wastewater treatment plants, superfund sites, and industrial runoff on microbial diversity. The research demonstrates the importance of understanding longitudinal sediment transport and deposition in estuarine systems under daily flow regimes but more critically, following natural hazard events to ensure sustainability and resilience of systems such as the GBES that encounter numerous acute and chronic stresses.

4.
Analyst ; 146(15): 4835-4840, 2021 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-34198311

RESUMO

We introduce analyte-dependent exclusion of reporter reagents from restricted-access adsorbents as the basis of an isocratic reporter-exclusion immunoassay for viruses, proteins, and other analytes. Capto™ Core 700 and related resins possess a noninteracting size-selective outer layer surrounding a high-capacity nonspecific mixed-mode capture adsorbent core. In the absence of analyte, antibody-enzyme reporter conjugates can enter the adsorbent and be captured, and their signal is lost. In the presence of large or artificially-expanded analytes, reporter reagents bind to analyte species to form complexes large enough to be excluded from the adsorbent core, allowing their signal to be observed. This assay principle is demonstrated using M13 bacteriophage virus and human chorionic gonadotropin as model analytes. The simple isocratic detection approach described here allows a rapid implementation of immunoassay for detection of a wide range of analytes and uses inexpensive, generally-applicable, and stable column materials instead of costly analyte-specific immunoaffinity adsorbents.


Assuntos
Bacteriófago M13 , Gonadotropina Coriônica , Humanos , Imunoensaio , Indicadores e Reagentes
5.
Angew Chem Weinheim Bergstr Ger ; 133(18): 10361-10366, 2021 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-34230707

RESUMO

The receptor-binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 spike (S) protein plays a central role in mediating the first step of virus infection to cause disease: virus binding to angiotensin-converting enzyme 2 (ACE2) receptors on human host cells. Therefore, S/RBD is an ideal target for blocking and neutralization therapies to prevent and treat coronavirus disease 2019 (COVID-19). Using a target-based selection approach, we developed oligonucleotide aptamers containing a conserved sequence motif that specifically targets S/RBD. Synthetic aptamers had high binding affinity for S/RBD-coated virus mimics (K D≈7 nM) and also blocked interaction of S/RBD with ACE2 receptors (IC50≈5 nM). Importantly, aptamers were able to neutralize S protein-expressing viral particles and prevent host cell infection, suggesting a promising COVID-19 therapy strategy.

6.
Angew Chem Int Ed Engl ; 60(18): 10273-10278, 2021 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-33684258

RESUMO

The receptor-binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 spike (S) protein plays a central role in mediating the first step of virus infection to cause disease: virus binding to angiotensin-converting enzyme 2 (ACE2) receptors on human host cells. Therefore, S/RBD is an ideal target for blocking and neutralization therapies to prevent and treat coronavirus disease 2019 (COVID-19). Using a target-based selection approach, we developed oligonucleotide aptamers containing a conserved sequence motif that specifically targets S/RBD. Synthetic aptamers had high binding affinity for S/RBD-coated virus mimics (KD ≈7 nM) and also blocked interaction of S/RBD with ACE2 receptors (IC50 ≈5 nM). Importantly, aptamers were able to neutralize S protein-expressing viral particles and prevent host cell infection, suggesting a promising COVID-19 therapy strategy.


Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , Antivirais/farmacologia , Aptâmeros de Nucleotídeos/farmacologia , COVID-19/tratamento farmacológico , SARS-CoV-2/efeitos dos fármacos , Glicoproteína da Espícula de Coronavírus/metabolismo , Antivirais/química , Aptâmeros de Nucleotídeos/química , Sequência de Bases , COVID-19/metabolismo , Células HEK293 , Humanos , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Mapas de Interação de Proteínas/efeitos dos fármacos , SARS-CoV-2/química , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/química
7.
Biosens Bioelectron ; 165: 112327, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32729475

RESUMO

Purification of therapeutic monoclonal antibodies usually involves a protein A affinity capture step. Because column breakthrough of antibody in complex, UV-absorbing culture fluid cannot be readily detected in real time, processes are designed so conservatively that column capacity is usually underutilized, wasting adsorbent and reducing productivity. We have developed a fluorescence-based monitoring technology which allows real-time mAb monitoring and used it to detect IgG in column breakthrough. The column effluent was continuously contacted with soluble fluorescein-labeled Fc-binding ligands to produce an immediately-detectable shift in both fluorescence polarization and intensity. To extend the upper limit of inlet flow rate, a 14:1 split-ratio flow splitter was tested with an inlet flow of 15 mL/min (0.9 L/h), producing a sampling stream at 1 mL/min while still enabling continuous detection functionality. We observed significant shifts in fluorescence intensity in CHO cell culture fluid spiked with human IgG, and detected 0.02-0.1 g/L human IgG in protein A column breakthrough at a flow velocity of 80 cm/h. The increase in fluorescence intensity upon 0.1% breakthrough of an 1 g/L feed was used to trigger column switching using Python-enabled two-way communication with the standard Unicorn OPC process control protocol. The technology allows rapid, continuous and reliable monitoring of IgG in a flowing process stream, without elaborate sample preparation.


Assuntos
Técnicas Biossensoriais , Proteína Estafilocócica A , Animais , Células CHO , Cromatografia de Afinidade , Cricetinae , Cricetulus , Humanos
8.
Anal Methods ; 12(3): 272-280, 2020 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-32577135

RESUMO

Incorporating two persistent luminescent nanophosphors (PLNPs), green-emitting SrAl2O4:Eu2+,Dy3+ (SAO) and blue-emitting (Sr0.625Ba0.375)2MgSi2O7:Eu2+,Dy3+ (SBMSO), in a single lateral flow assay (LFA) establishes a luminescence-based, multiplex point-of-need test capable of simultaneously detecting two different analytes in a single sample. The advantages of this system are the high sensitivity and photostability of PLNPs, while only requiring access to minimal hardware and a smartphone for signal detection. The PLNPs were obtained by first wet milling bulk synthesized phosphor powders, followed by fractionation using differential centrifugal sedimentation to obtain monodisperse nanoparticles. A modified Stöber process was then employed to encapsulate the nanoparticles in a water-stable silica shell followed by attaching antibodies to the particles' surfaces using reductive amination chemistry. The resulting PLNPs were incorporated in an LFA to concurrently detect two independent model analytes, prostate-specific antigen (PSA) and human chorionic gonadotropin (hCG). The multicolor-multiplex PLNP-based assays were finally imaged using a smartphone-based imaging system with excellent detection limits (0.1 ng mL-1 of PSA and 1 ng mL-1 of hCG) that are competitive with commercially available LFAs.


Assuntos
Luminescência , Nanopartículas , Bioensaio , Humanos , Dióxido de Silício
9.
Analyst ; 145(14): 4942-4949, 2020 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-32500871

RESUMO

We have developed an immuno-PCR based diagnostic platform which couples detection antibodies to self-assembled, ultra-detectable DNA-avidin nanoparticles stabilized with poly(ethylene glycol) to link DNA amplification to target protein concentration. Electrostatic neutralization and cloaking of the PCR-amplifiable DNA labels by avidin and PEG coating reduces non-specific "stickiness" and enhances assay sensitivity. We further optimized the detectability of the nanoparticles by incorporating four repeats of a unique synthetic DNA PCR target into each nanoparticle. Using human chorionic gonadotropin hormone (hCG) as a model analyte, this platform was able to quantitate the target hCG protein in femtomolar concentrations using only standard laboratory equipment.


Assuntos
Avidina , Nanopartículas , Anticorpos , DNA/genética , Humanos , Reação em Cadeia da Polimerase
10.
Biotechnol Bioeng ; 117(7): 2100-2115, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32255523

RESUMO

Biopharmaceutical product and process development do not yet take advantage of predictive computational modeling to nearly the degree seen in industries based on smaller molecules. To assess and advance progress in this area, spirited coopetition (mutually beneficial collaboration between competitors) was successfully used to motivate industrial scientists to develop, share, and compare data and methods which would normally have remained confidential. The first "Highland Games" competition was held in conjunction with the October 2018 Recovery of Biological Products Conference in Ashville, NC, with the goal of benchmarking and assessment of the ability to predict development-related properties of six antibodies from their amino acid sequences alone. Predictions included purification-influencing properties such as isoelectric point and protein A elution pH, and biophysical properties such as stability and viscosity at very high concentrations. Essential contributions were made by a large variety of individuals, including companies which consented to provide antibody amino acid sequences and test materials, volunteers who undertook the preparation and experimental characterization of these materials, and prediction teams who attempted to predict antibody properties from sequence alone. Best practices were identified and shared, and areas in which the community excels at making predictions were identified, as well as areas presenting opportunities for considerable improvement. Predictions of isoelectric point and protein A elution pH were especially good with all-prediction average errors of 0.2 and 1.6 pH unit, respectively, while predictions of some other properties were notably less good. This manuscript presents the events, methods, and results of the competition, and can serve as a tutorial and as a reference for in-house benchmarking by others. Organizations vary in their policies concerning disclosure of methods, but most managements were very cooperative with the Highland Games exercise, and considerable insight into common and best practices is available from the contributed methods. The accumulated data set will serve as a benchmarking tool for further development of in silico prediction tools.


Assuntos
Anticorpos Monoclonais/química , Produtos Biológicos/química , Descoberta de Drogas/métodos , Sequência de Aminoácidos , Humanos , Rituximab/química
11.
Sci Rep ; 10(1): 5078, 2020 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-32193476

RESUMO

Systemic anaplastic large cell lymphoma (ALCL) is an aggressive T-cell lymphoma most commonly seen in children and young adults. The majority of pediatric ALCLs are associated with the t(2;5)(p23;q35) translocation which fuses the Anaplastic Lymphoma Kinase (ALK) gene with the Nucleophosmin (NPM) gene. The NPM-ALK fusion protein is a constitutively-active tyrosine kinase, and plays a major role in tumor pathogenesis. In an effort to advance novel diagnostic approaches and the understanding of the function of this fusion protein in cancer cells, we expressed in E. coli, purified and characterized human NPM-ALK fusion protein to be used as a standard for estimating expression levels in cultured human ALCL cells, a key tool in ALCL pathobiology research. We estimated that NPM-ALK fusion protein is expressed at substantial levels in both Karpas 299 and SU-DHL-1 cells (ca. 4-6 million molecules or 0.5-0.7 pg protein per cell; based on our in-house developed NPM-ALK ELISA; LOD of 40 pM) as compared to the ubiquitous ß-actin protein (ca. 64 million molecules or 4.5 pg per lymphocyte). We also compared NPM-ALK/ ß-actin ratios determined by ELISA to those independently determined by two-dimensional electrophoresis and showed that the two methods are in good agreement.


Assuntos
Expressão Gênica , Linfoma Anaplásico de Células Grandes/genética , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Recombinação Genética/genética , Actinas/genética , Actinas/metabolismo , Adolescente , Linhagem Celular Tumoral , Criança , Eletroforese em Gel Bidimensional , Ensaio de Imunoadsorção Enzimática , Humanos , Proteínas Tirosina Quinases/fisiologia , Translocação Genética/genética , Adulto Jovem
12.
Sensors (Basel) ; 19(24)2019 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-31835468

RESUMO

This work presents a proof-of-concept demonstration of a novel inductive transducer, the femtoMag, that can be integrated with a lateral-flow assay (LFA) to provide detection and quantification of molecular biomarkers. The femtoMag transducer is manufactured using a low-cost printed circuit board (PCB) technology and can be controlled by relatively inexpensive electronics. It allows rapid high-precision quantification of the number (or amount) of superparamagnetic nanoparticle reporters along the length of an LFA test strip. It has a detection limit of 10-10 emu, which is equivalent to detecting 4 ng of superparamagnetic iron oxide (Fe3O4) nanoparticles. The femtoMag was used to quantify the hCG pregnancy hormone by quantifying the number of 200 nm magnetic reporters (superparamagnetic Fe3O4 nanoparticles embedded into a polymer matrix) immuno-captured within the test line of the LFA strip. A sensitivity of 100 pg/mL has been demonstrated. Upon further design and control electronics improvements, the sensitivity is projected to be better than 10 pg/mL. Analysis suggests that an average of 109 hCG molecules are needed to specifically bind 107 nanoparticles in the test line. The ratio of the number of hCG molecules in the sample to the number of reporters in the test line increases monotonically from 20 to 500 as the hCG concentration increases from 0.1 ng/mL to 10 ng/mL. The low-cost easy-to-use femtoMag platform offers high-sensitivity/high-precision target analyte quantification and promises to bring state-of-the-art medical diagnostic tests to the point of care.

13.
PLoS One ; 14(12): e0225365, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31821330

RESUMO

Herpes Simplex Virus Type 2 (HSV-2) is a common human pathogen that causes life-long illness. The US prevalence of HSV-2 infection is 11.9% for individuals between 15 and 49 years of age. Individuals with HSV-2 infection are more likely to contract and spread other sexually-transmitted infections. Eighty percent of individuals with HSV-2 are unaware of their infection, in part because of the social stigma associated with in-clinic testing for sexually-transmitted infections. We conducted an initial evaluation of a prototype smartphone-based serological lateral-flow immunoassay (LFA) for HSV-2 infection that uses strontium aluminate persistent luminescent nanoparticles (nanophosphors) as reporters. When applied to a test panel of 21 human plasma/serum samples varying in anti-HSV titer, the nanophosphor HSV-2 LFA had 96.7% sensitivity and 100% specificity for detection of HSV-2 infection. The sensitivity of the nanophosphor HSV-2 LFA was higher than that of commercially-available rapid HSV-2 assays tested with the same panel. Analysis of the iPhone nanophosphor HSV-2 LFA strip images with our custom smartphone app gave greater reproducibility compared to ImageJ analysis of strip images. The smartphone-based nanophosphor LFA technology shows promise for private self-testing for sexually-transmitted infections (STI).


Assuntos
Herpes Simples/diagnóstico , Herpesvirus Humano 2/imunologia , Imunoensaio , Autoavaliação Diagnóstica , Humanos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
16.
ACS Appl Mater Interfaces ; 10(38): 31845-31849, 2018 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-30168312

RESUMO

Proximity ligation assay (PLA) achieves extremely low limits of detection but requires the synthesis of antibody-DNA conjugates recognizing multiple target epitopes with appropriate proximity. In this work, we introduce a more generally applicable method by replacing antibody-DNA conjugates with nanoparticles which create ultradetectable PCR templates by capturing biotinylated oligonucleotides and catalyzing ligation. A competitive PCR readout was used to make the assay quantitative. We have demonstrated that NP-PLA can detect and quantitate human chorionic gonadotropin (hCG) levels as low as 2.6 fM (∼0.1 pg/mL), nearly 1000 times more sensitive than the current state of the art ELISA.


Assuntos
Bioensaio/métodos , Biomarcadores/análise , Nanopartículas/química , Proteínas/análise , Gonadotropina Coriônica/sangue , Ensaio de Imunoadsorção Enzimática/normas , Humanos , Reação em Cadeia da Polimerase
17.
Artigo em Inglês | MEDLINE | ID: mdl-30050871

RESUMO

Changes in the population levels of specific bacterial species within the gut microbiome have been linked to a variety of illnesses. Most assays that determine the relative abundance of specific taxa are based on amplification and sequencing of stable phylogenetic gene regions. Such lab-based analysis requires pre-analytical sample preservation and storage that have been shown to introduce biases in the characterization of microbial profiles. Recombinase polymerase amplification (RPA) is an isothermal nucleic acid amplification method that employs commercially available, easy-to-use freeze-dried enzyme pellets that can be used to analyze specimens rapidly in the field or clinic, using a portable fluorometer. Immediate analysis of diverse bacterial communities can lead to a more accurate quantification of relative bacterial abundance. In this study, we discovered that universal bacterial 16S ribosomal DNA primers give false-positive signals in RPA analysis because manufacturing host Escherichia coli DNA is present in the RPA reagents. The manufacturer of RPA reagents advises against developing an RPA assay that detects the presence of E. coli due to the presence of contaminating E. coli DNA in the reaction buffer (www.twistdx.co.uk/). We, therefore, explored four strategies to deplete or fragment extraneous DNA in RPA reagents while preserving enzyme activity: metal-chelate affinity chromatography, sonication, DNA cleavage using methylation-dependent restriction endonucleases, and DNA depletion using anti-DNA antibodies. Removing DNA with anti-DNA antibodies enabled the development of a quantitative RPA microbiome assay capable of determining the relative abundance of the physiologically-important bacterium Akkermansia muciniphila in human feces.


Assuntos
Carga Bacteriana/métodos , Reações Falso-Positivas , Fezes/microbiologia , Microbiota , Técnicas de Amplificação de Ácido Nucleico/métodos , Verrucomicrobia/isolamento & purificação , DNA Bacteriano/análise , DNA Bacteriano/genética , DNA Ribossômico/análise , DNA Ribossômico/genética , Humanos , RNA Ribossômico 16S/genética
18.
Langmuir ; 34(12): 3694-3700, 2018 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-29509429

RESUMO

Maintaining colloidal stability in unfriendly environments while retaining surface chemical properties is challenging for fundamental science and crucial for many applications. Here, we report for the first time that by using a low concentration of poly(sodium 4-styrenesulfonate) (PSS), graphene-based amphiphilic Janus nanosheets (AJNs) can be stabilized in high salt brine (3 wt % NaCl and 0.5 wt % CaCl2), whereas the interfacial behavior of the nanosheets is not affected. The adsorption of PSS on the hydrophilic and hydrophobic surfaces of AJNs in brine was investigated experimentally and by molecular dynamics simulations. Simulations further showed that the spatial configuration of absorbed PSS molecules with sulfonate functional groups facing outward favored the generation of electrosteric repulsive interactions. Calculations of the interaction energy between PSS molecules and the nanosheet revealed surface charge as a key parameter to stabilize AJNs in the salt environment, as demonstrated by the case of graphene oxide with higher surface charge. Simulations were also used to examine the interfacial behavior of graphene-based AJNs in biphasic systems. The AJNs, which exhibited asymmetry in surface wettability, remained at the oil/brine interface because of PSS detachment from the hydrophobic surface. The results were subsequently experimentally confirmed, consistent with our previously reported graphene-based AJN fluid prepared in fresh water. The process was thermodynamically supported by the demonstrated negative change of Gibbs free energy. We believe that such a strategy could benefit for the stabilization of other AJNs with surface chemical accessibility under harsh conditions.

19.
PLoS One ; 13(1): e0186782, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29309424

RESUMO

Lateral flow assays (LFAs) are a widely-used point-of care diagnostic format, but suffer from limited analytical sensitivity, especially when read by eye. It has recently been reported that LFA performance can be improved by using magnetic reporter particles and an external magnetic field applied at the test line. The mechanism of sensitivity/performance enhancement was suggested to be concentration/retardation of reporter particles at the test line. Here we demonstrate an additional mechanism of particle relocation where reporter particles from the lower depths of the translucent LFA strip relocate to more-visible locations nearer to the top surface, producing a more visible signal. With a magnetic field we observed an improvement in sensitivity of human chorionic gonadotropin (hCG) detection from 1.25 ng/mL to 0.31 ng/mL. We also observed an increase of the color intensity per particle in test lines when the magnetic field was present.


Assuntos
Campos Eletromagnéticos , Anticorpos/imunologia , Gonadotropina Coriônica/análise , Gonadotropina Coriônica/imunologia , Limite de Detecção , Soroalbumina Bovina/análise
20.
J Chromatogr A ; 1532: 246-250, 2018 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-29224842

RESUMO

M13 is a filamentous, non-lytic bacteriophage that infects Escherichia coli via the F pilus. Currently, phage M13 is widely used in phage display technology and bio-nanotechnology, and is considered a possible antibacterial therapeutic agent, among other applications. Conventional phage purification involves 5-7 operational steps, with high operational costs and significant product loss (approximately 60%). In this work, we propose a scalable purification process for M13 bacteriophage using a novel stationary phase based on a polymeric ionic liquid (PIL) with a positively charged backbone structure. Poly (1-vinyl-3-ethyl imidazolium bis(trifluoromethylsulfonyl) imide) - poly(VEIM-TFSI) predominantly acted as an anion exchanger under binding-elution mode. This revealed to be a rapid and simple method for the recovery of phage M13 with an overall separation yield of over 70% after a single downstream step. To the best of our knowledge, PILs have never been used as separation matrices for biological products and the results obtained, together with the large number of cations and anions available to prepare PILs, illustrate well the large potential of the proposed methodology.


Assuntos
Bacteriófago M13/isolamento & purificação , Líquidos Iônicos/química , Adsorção , Ânions , Tampões (Química) , Polímeros/química
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