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1.
Mikrochim Acta ; 191(9): 522, 2024 08 07.
Artículo en Inglés | MEDLINE | ID: mdl-39112842

RESUMEN

An ultrasensitive photothermal assay was designed for point-of-care testing (POCT) of tumor markers based on a filter membrane. Firstly, Cu2-xSe was successfully encapsulated in liposome spheres with biotin on the surface and connected to carcinoembryonic antigen (CEA) aptamer with 3'end modified biotin by streptavidin. Secondly, the CEA antibody was successfully modified on the surface of the nitrocellulose membrane through simple incubation. Finally, the assay process was completed using a disposable syringe, and the temperature was recorded using a handheld infrared temperature detector. In the range 0-50 ng mL-1, the temperature change of the nitrocellulose membrane has a strong linear relationship with CEA concentration, and the detection limit is 0.097 ng mL-1. It is worth noting that the entire testing process can be easily performed in 10 min, much shorter than traditional clinical methods. In addition, this method was successfully applied to the quantitative determination of CEA levels in human serum samples with a recovery of 96.2-103.3%. This rapid assay can be performed by "one suction and one push" through a disposable syringe, which is simple to operate, and the excellent sensitivity reveals the great potential of the proposed strategy in the POCT of tumor biomarkers.


Asunto(s)
Aptámeros de Nucleótidos , Biomarcadores de Tumor , Antígeno Carcinoembrionario , Cobre , Límite de Detección , Humanos , Antígeno Carcinoembrionario/sangre , Cobre/química , Aptámeros de Nucleótidos/química , Biomarcadores de Tumor/sangre , Liposomas/química , Técnicas Biosensibles/métodos , Sistemas de Atención de Punto , Temperatura , Biotina/química , Pruebas en el Punto de Atención , Colodión/química
2.
Biosens Bioelectron ; 262: 116544, 2024 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-38963952

RESUMEN

In this work, a nanostructured conductive film possessing nanozyme features was straightforwardly produced via laser-assembling and integrated into complete nitrocellulose sensors; the cellulosic substrate allows to host live cells, while the nanostructured film nanozyme activity ensures the enzyme-free real-time detection of hydrogen peroxide (H2O2) released by the sames. In detail, a highly exfoliated reduced graphene oxide 3D film decorated with naked platinum nanocubes was produced using a CO2-laser plotter via the simultaneous reduction and patterning of graphene oxide and platinum cations; the nanostructured film was integrated into a nitrocellulose substrate and the complete sensor was manufactured using an affordable semi-automatic printing approach. The linear range for the direct H2O2 determination was 0.5-80 µM (R2 = 0.9943), with a limit of detection of 0.2 µM. Live cell measurements were achieved by placing the sensor in the culture medium, ensuring their adhesion on the sensors' surface; two cell lines were used as non-tumorigenic (Vero cells) and tumorigenic (SKBR3 cells) models, respectively. Real-time detection of H2O2 released by cells upon stimulation with phorbol ester was carried out; the nitrocellulose sensor returned on-site and real-time quantitative information on the H2O2 released proving useful sensitivity and selectivity, allowing to distinguish tumorigenic cells. The proposed strategy allows low-cost in-series semi-automatic production of paper-based point-of-care devices using simple benchtop instrumentation, paving the way for the easy and affordable monitoring of the cytopathology state of cancer cells.


Asunto(s)
Técnicas Biosensibles , Colodión , Grafito , Peróxido de Hidrógeno , Nanoestructuras , Peróxido de Hidrógeno/análisis , Humanos , Técnicas Biosensibles/instrumentación , Grafito/química , Nanoestructuras/química , Colodión/química , Línea Celular Tumoral , Rayos Láser , Animales , Platino (Metal)/química , Neoplasias , Límite de Detección
3.
Biosens Bioelectron ; 258: 116315, 2024 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-38701536

RESUMEN

Research in electrochemical detection in lateral flow assays (LFAs) has gained significant momentum in recent years. The primary impetus for this surge in interest is the pursuit of achieving lower limits of detection, especially given that LFAs are the most widely employed point-of-care biosensors. Conventionally, the strategy for merging electrochemistry and LFAs has centered on the superposition of screen-printed electrodes onto nitrocellulose substrates during LFA fabrication. Nevertheless, this approach poses substantial limitations regarding scalability. In response, we have developed a novel method for the complete integration of reduced graphene oxide (rGO) electrodes into LFA strips. We employed a CO2 laser to concurrently reduce graphene oxide and pattern nitrocellulose, exposing its backing to create connection sites impervious to sample leakage. Subsequently, rGO and nitrocellulose were juxtaposed and introduced into a roll-to-roll system using a wax printer. The exerted pressure facilitated the transfer of rGO onto the nitrocellulose. We systematically evaluated several electrochemical strategies to harness the synergy between rGO and LFAs. While certain challenges persist, our rGO transfer technology presents compelling potential for setting a new standard in electrochemical LFA fabrication.


Asunto(s)
Técnicas Biosensibles , Técnicas Electroquímicas , Electrodos , Grafito , Sistemas de Atención de Punto , Grafito/química , Técnicas Biosensibles/instrumentación , Técnicas Biosensibles/métodos , Técnicas Electroquímicas/métodos , Técnicas Electroquímicas/instrumentación , Humanos , Diseño de Equipo , Colodión/química , Pruebas en el Punto de Atención , Límite de Detección , Oxidación-Reducción
4.
Biosens Bioelectron ; 258: 116376, 2024 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-38739999

RESUMEN

The capacitive immunosensor, known for its label-free simplicity, has great potential for point-of-care diagnostics. However, the interaction between insulation and recognition layers on the sensing electrode greatly affects its performance. This study introduces a pioneering dual-layer strategy, implementing a novel combination of acrylic resin (AR) and nitrocellulose (NC) coatings on screen-printed carbon electrodes (SPCEs). This innovative approach not only enhances the dielectric properties of the capacitive sensor but also streamlines the immobilization of recognizing elements. Particularly noteworthy is the superior reliability and insulation offered by the AR coating, surpassing the limitations of traditional self-assembled monolayer (SAM) modifications. This dual-layer methodology establishes a robust foundation for constructing capacitive sensors optimized specifically for liquid medium-based biosensing applications. The NC coating in this study represents a breakthrough in effectively immobilizing BSA, unraveling the capacitive response intricately linked to the quantity of adsorbed recognizing elements. The results underscore the prowess of the proposed immunosensor, showcasing a meticulously defined linear calibration curve for anti-BSA (ranging from 0 to 25 µg/ml). Additionally, specific interactions with anti-HAS and anti-TNF-α further validate the versatility and efficacy of the developed immunosensor. This work presents a streamlined and highly efficient protocol for developing label-free immunosensors for antibody determination and introduces a paradigm shift by utilizing readily available electrodes and sensing systems. The findings are poised to catalyze a significant acceleration in the advancement of biosensor technology, opening new avenues for innovative applications in point-of-care diagnostics.


Asunto(s)
Resinas Acrílicas , Técnicas Biosensibles , Carbono , Colodión , Electrodos , Albúmina Sérica Bovina , Técnicas Biosensibles/instrumentación , Carbono/química , Resinas Acrílicas/química , Inmunoensayo/instrumentación , Inmunoensayo/métodos , Colodión/química , Albúmina Sérica Bovina/química , Humanos , Capacidad Eléctrica , Límite de Detección , Técnicas Electroquímicas/métodos , Anticuerpos Inmovilizados/química , Animales
5.
Anal Biochem ; 692: 115575, 2024 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-38796117

RESUMEN

This study demonstrates, for the first time, the proof-of-concept of a novel immunosensor, a touchpad-based immunochromatographic strip, that non-invasively extracts and detects skin surface proteins. The strip was composed of a nitrocellulose membrane at the center, where a spot of anti-human IgG capture antibody was physically adsorbed. The capture antibody spot was covered with a glass fiber membrane impregnated with phosphate-buffered saline (PBS) to extract skin surface proteins, avoiding direct contact of the human skin with the capture antibodies. Skin surface IgG was detected in two steps: (1) touching the capture antibody via a glass fiber membrane containing PBS, and (2) dipping the strip into the Au-nanoparticle-labeled secondary antibody to visualize the existence of the captured skin surface IgG on the strip. We qualitatively demonstrated that using a very small amount of PBS while maintaining contact with the skin, skin surface proteins can be concentrated and detected, even with a relatively low-sensitivity immunochromatographic chip. This sensor is expected to be a potential biosensor for the non-invasive diagnosis of the integrity of human skin.


Asunto(s)
Cromatografía de Afinidad , Piel , Humanos , Piel/química , Cromatografía de Afinidad/métodos , Oro/química , Proteínas de la Membrana/análisis , Proteínas de la Membrana/inmunología , Inmunoglobulina G/análisis , Inmunoglobulina G/inmunología , Nanopartículas del Metal/química , Colodión/química , Técnicas Biosensibles/métodos
6.
Talanta ; 274: 126007, 2024 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-38583331

RESUMEN

Hypoxanthine (Hx), produced by adenosine triphosphate (ATP) metabolism, is a valuable indicator that determines the quality and degradation status of meat products and is also an important biochemical marker to certain diseases such as gout. The rapid emergence of paper-based enzyme biosensors has already revolutionized its on-site determination. But it is still limited by the complex patterning and fabrication, unstable enzyme and uneven coloration. This work aims to develop an eco-friendly method to construct engineered paper microfluidic, which seeks to produce reaction and non-reaction zones without any patterning procedure. Chito-oligosaccharide (COS), derived from shrimp shells, was used to modify nitrocellulose membranes and immobilize xanthine oxidase (XOD) and chromogenic agent of nitro blue tetrazolium chloride (NBT). After modification, micro fluids could converge into the modification area and Hx could be detected by XOD-catalyzed conversion. Due to the positively charged cationic basic properties of COS, the enzyme storage stability and the color homogeneity could be greatly strengthened through the electrostatic attraction between COS and XOD and formazan product. The detection limit (LOD) is 2.30 µM; the linear range is 0.05-0.35 mM; the complete test time can be as short as 5 min. The COS-based biosensor shows high specificity and can be used directly for Hx in complex samples such as fish and shrimp samples, and different broths. This biosensor is eco-friendly, nontechnical, economical and therefore a compelling platform for on-site or home-based detection of food freshness.


Asunto(s)
Técnicas Biosensibles , Colodión , Hipoxantina , Oligosacáridos , Xantina Oxidasa , Animales , Oligosacáridos/química , Oligosacáridos/análisis , Técnicas Biosensibles/métodos , Hipoxantina/análisis , Hipoxantina/química , Colodión/química , Xantina Oxidasa/química , Xantina Oxidasa/metabolismo , Peces , Quitina/química , Enzimas Inmovilizadas/química , Enzimas Inmovilizadas/metabolismo , Tecnología Química Verde/métodos , Propiedades de Superficie , Límite de Detección
7.
Anal Methods ; 16(16): 2489-2495, 2024 04 25.
Artículo en Inglés | MEDLINE | ID: mdl-38502566

RESUMEN

Iron deficiency anemia (IDA) is a condition characterized by lower-than-average iron (Fe) levels in the body, affecting a substantial number of young children and pregnant women globally. Existing diagnostic methods for IDA rely on invasive analysis of stored Fe in ferritin from blood samples, posing challenges, especially for toddlers and young children. To address this issue, saliva has been proposed as a non-invasive sample matrix for IDA diagnosis. However, conventional Fe analysis techniques often necessitate complex and costly instrumentation. This study presents the first non-invasive, saliva-based preliminary screening test for IDA using a nitrocellulose lateral flow system. In this study, we introduce a novel approach using the ferroin reaction with bathophenanthroline (Bphen) and ferrous (Fe2+) ions to quantify Fe levels in saliva. Our methodology involves a capillary flow-driven microfluidic device integrated into a lateral flow system utilizing nitrocellulose membranes. Here, we present the first instance of saliva on a nitrocellulose substrate to detect salivary Fe levels. The optimized system yielded a linear response over the 1-200 ppm range in buffer solution, with a limit of detection (LoD) of 5.6 ppm. Furthermore, the system demonstrated a linear response in pooled saliva samples across the 1-1000 ppm range, with a LoD of 55.1 ppm. These results underscore the potential of our capillary flow-driven microfluidic device as a viable non-invasive diagnostic tool for IDA, particularly in remote and resource-limited settings.


Asunto(s)
Anemia Ferropénica , Hierro , Saliva , Humanos , Saliva/química , Anemia Ferropénica/diagnóstico , Hierro/análisis , Femenino , Límite de Detección , Técnicas Analíticas Microfluídicas/instrumentación , Técnicas Analíticas Microfluídicas/métodos , Colodión/química , Dispositivos Laboratorio en un Chip
8.
Biotechnol J ; 18(9): e2300143, 2023 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-37222181

RESUMEN

A paper microfluidic device capable of conducting enzyme-linked assays is presented: a microfluidic enzyme-linked paper analytical device (µEL-PAD). The system exploits a wash-free sandwich coupling to form beads/analyte/enzyme complexes, which are subsequently added to the vertical flow device composed of wax-printed paper, waxed nitrocellulose membrane and absorbent/barrier layers. The nitrocellulose retains the bead complexes without disrupting the flow, enabling for an efficient washing step. The entrapped complexes then interact with the chromogenic substrate stored on the detection paper, generating a color change on it, quantified with an open-source smartphone software. This is a universal paper-based technology suitable for high-sensitivity quantification of many analytes, such as proteins or nucleic acids, with different enzyme-linked formats. Here, the potential of the µEL-PAD is demonstrated to detect DNA from Staphylococcus epidermidis. After generation of isothermally amplified genomic DNA from bacteria, Biotin/FITC-labeled products were analyzed with the µEL-PAD, exploiting streptavidin-coated beads and antiFITC-horseradish peroxidase. The µEL-PAD achieved a limit of detection (LOD) and quantification <10 genome copies/µL, these being at least 70- and 1000-fold lower, respectively, than a traditional lateral flow assay (LFA) exploiting immobilized streptavidin and antiFITC-gold nanoparticles. It is envisaged that the device will be a good option for low-cost, simple, quantitative, and sensitive paper-based point-of-care testing.


Asunto(s)
Técnicas de Química Analítica , Microfluídica , Papel , Microfluídica/instrumentación , Colodión/química , Compuestos Cromogénicos/química , Aplicaciones Móviles , Proteínas/análisis , Ácidos Nucleicos/análisis , Límite de Detección , Técnicas de Química Analítica/instrumentación , Técnicas de Química Analítica/métodos
9.
ACS Appl Mater Interfaces ; 15(5): 6561-6571, 2023 Feb 08.
Artículo en Inglés | MEDLINE | ID: mdl-36692231

RESUMEN

Paper-based platforms are ideal for on-site surveillance of infectious diseases in low-resource settings due to their simplicity, self-containment, and low cost. The two most popular materials used in paper-based platforms are nitrocellulose and cellulose. The nitrocellulose membrane has a high protein binding affinity, but its high price is an issue. Cellulose paper is inexpensive and allows intricate fluidic control for more sophisticated biochemical reactions, but it has a low protein binding affinity. By examining the microstructure of cellulose paper, we discover that cellulose fibers in the paper matrix are covered by thin films, which possibly result from the additives used in the paper-making process. Our finding suggests that the thin films are inert to protein adsorption. By selectively depleting the inert films with reactive plasma, we were able to enhance the protein adsorption to the cellulose paper and improve the performance of lateral flow assays. The performance of certain lateral flow assays on the plasma-treated cellulose paper is equivalent to or better than that on the nitrocellulose membrane. This leads us to believe that cellulose paper with a microstructure exclusively designed for protein binding, either by refined paper manufacturing process or by post-manufacture modification such as the plasma treatment presented herein, can potentially replace nitrocellulose as a less expensive paper substrate for point-of-care rapid test kits.


Asunto(s)
Celulosa , Proteínas , Celulosa/química , Colodión/química , Adsorción , Proteínas/química , Unión Proteica
10.
Molecules ; 27(20)2022 Oct 17.
Artículo en Inglés | MEDLINE | ID: mdl-36296538

RESUMEN

This research aims to develop new high-energy dense ordinary- and nano-energetic composites based on hydrazine 3-nitro-1,2,4-triazol-5-one (HNTO) and nitrated cellulose and nanostructured nitrocellulose (NC and NMCC). The elaborated energetic formulations (HNTO/NC and HNTO/NMCC) were fully characterized in terms of their chemical compatibility, morphology, thermal stability, and energetic performance. The experimental findings implied that the designed HNTO/NC and HNTO/NMCC formulations have good compatibilities with attractive characteristics such as density greater than 1.780 g/cm3 and impact sensitivity around 6 J. Furthermore, theoretical performance calculations (EXPLO5 V6.04) displayed that the optimal composition of the as-prepared energetic composites yielded excellent specific impulses and detonation velocities, which increased from 205.7 s and 7908 m/s for HNTO/NC to 209.6 s and 8064 m/s for HNTO/NMCC. Moreover, deep insight on the multi-step kinetic behaviors of the as-prepared formulations was provided based on the measured DSC data combined with isoconversional kinetic methods. It is revealed that both energetic composites undergo three consecutive exothermic events with satisfactory activation energies in the range of 139-166 kJ/mol for HNTO/NC and 119-134 kJ/mol for HNTO/NMCC. Overall, this research displayed that the new developed nanoenergetic composite based on nitrated cellulose nanostructure could serve as a promising candidate for practical applications in solid rocket propellants and composite explosives.


Asunto(s)
Hidrazinas , Nanoestructuras , Colodión/química , Cinética
11.
Anal Sci ; 38(11): 1417-1424, 2022 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-35932412

RESUMEN

An accurate, simple and safe method was developed for simultaneous determination of nitroglycerine (NG) and nitrocellulose (NC) in double base solid propellants (DB propellants). The proposed method is based on alkaline hydrolysis of NG and NC, and followed by colored reaction of released nitrite ion with p-nitroaniline in the presence of diphenylamine in acidic media and produce azo dye. The absorbance of the azo dye was measured at 534 nm. Two sets of reaction conditions were developed. In the first set, at room temperature, only NG was hydrolyzed and calibration curve obtained. In the second set, at 60 â„ƒ, NG and NC were hydrolyzed simultaneously. Based on obtained amount for the NG at room temperature, and total amount of NG and NC at 60 â„ƒ, the amount of NC was determined by using stoichiometric equations. The calibration curve was linear over the concentration ranges of 0.2-5.0, 0.5-10 µg mL-1 for NG and NC, respectively. The proposed method was successfully applied for the determination of NG and NC in DB propellants with good recoveries ranged from 99 to 101%, and RSD less than 2.0%. The method statistically compared based on t- and F-tests with those obtained in according to military standard method (MIL-STD-286). The results showed that the proposed method offers an accuracy and reliable approach for the determination of these compounds in DB propellants, and can be suggested as a routine method in military quality control laboratories.


Asunto(s)
Nitritos , Nitroglicerina , Colodión/química , Nitritos/química , Difenilamina , Compuestos Azo/química
12.
ACS Appl Mater Interfaces ; 13(35): 41445-41453, 2021 Sep 08.
Artículo en Inglés | MEDLINE | ID: mdl-34428374

RESUMEN

Airborne transmission of exhaled virus can rapidly spread, thereby increasing disease progression from local incidents to pandemics. Due to the COVID-19 pandemic, states and local governments have enforced the use of protective masks in public and work areas to minimize the disease spread. Here, we have leveraged the function of protective face coverings toward COVID-19 diagnosis. We developed a user-friendly, affordable, and wearable collector. This noninvasive platform is integrated into protective masks toward collecting airborne virus in the exhaled breath over the wearing period. A viral sample was sprayed into the collector to model airborne dispersion, and then the enriched pathogen was extracted from the collector for further analytical evaluation. To validate this design, qualitative colorimetric loop-mediated isothermal amplification, quantitative reverse transcription polymerase chain reaction, and antibody-based dot blot assays were performed to detect the presence of SARS-CoV-2. We envision that this platform will facilitate sampling of current SARS-CoV-2 and is potentially broadly applicable to other airborne diseases for future emerging pandemics.


Asunto(s)
Pruebas Respiratorias/instrumentación , Prueba de COVID-19/instrumentación , Máscaras , SARS-CoV-2/aislamiento & purificación , Microbiología del Aire , Anticuerpos Antivirales/inmunología , Pruebas Respiratorias/métodos , Prueba de COVID-19/métodos , Colodión/química , Colorimetría/métodos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , Cemento de Policarboxilato/química , Porosidad , Prueba de Estudio Conceptual , ARN Viral/análisis , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , SARS-CoV-2/química , Proteínas Virales/análisis , Proteínas Virales/inmunología
13.
Carbohydr Polym ; 268: 118259, 2021 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-34127229

RESUMEN

Nitrocellulose (NC) membrane can have value-added applications for lateral flow assay (LFA)-based diagnostic tools, which has great potential for the detection of pathogens, such as COVID-19, in different environments. However, poor sensitivity of the NC membrane based LFA limits its further application in many cases. Herein, we developed a facile method for LFA sensitivity enhancement, by incorporating two-sugar barrier into LFAs: one between the conjugation pad and the test line, and the other between the test line and the control line. ORF1ab nucleic acid of COVID-19 was used as the model target to demonstrate the concept on the HF120 membrane. Results show that at optimum conditions, the two sugar barrier LFAs have a detection limit of 0.5 nM, which is compared to that of 2.5 nM for the control LFA, achieving a 5-fold sensitivity increase. This low cost, easy-to-fabricate and easy-to-integrate LFA method may have potential applications in other cellulose paper-based platforms.


Asunto(s)
Prueba de Ácido Nucleico para COVID-19/métodos , Colodión/química , ARN Mensajero/análisis , Azúcares/química , Proteínas Virales/genética , Prueba de Ácido Nucleico para COVID-19/instrumentación , ADN/química , Sondas de ADN/química , Oro/química , Límite de Detección , Nanopartículas del Metal/química , Poliproteínas/genética , SARS-CoV-2/química , Sensibilidad y Especificidad
14.
Sci Rep ; 11(1): 7880, 2021 04 12.
Artículo en Inglés | MEDLINE | ID: mdl-33846482

RESUMEN

Since the pandemic outbreak of Covid-19 in December 2019, several lateral flow assay (LFA) devices were developed to enable the constant monitoring of regional and global infection processes. Additionally, innumerable lateral flow test devices are frequently used for determination of different clinical parameters, food safety, and environmental factors. Since common LFAs rely on non-biodegradable nitrocellulose membranes, we focused on their replacement by cellulose-composed, biodegradable papers. We report the development of cellulose paper-based lateral flow immunoassays using a carbohydrate-binding module-fused to detection antibodies. Studies regarding the protein binding capacity and potential protein wash-off effects on cellulose paper demonstrated a 2.7-fold protein binding capacity of CBM-fused antibody fragments compared to the sole antibody fragment. Furthermore, this strategy improved the spatial retention of CBM-fused detection antibodies to the test area, which resulted in an enhanced sensitivity and improved overall LFA-performance compared to the naked detection antibody. CBM-assisted antibodies were validated by implementation into two model lateral flow test devices (pregnancy detection and the detection of SARS-CoV-2 specific antibodies). The CBM-assisted pregnancy LFA demonstrated sensitive detection of human gonadotropin (hCG) in synthetic urine and the CBM-assisted Covid-19 antibody LFA was able to detect SARS-CoV-2 specific antibodies present in serum. Our findings pave the way to the more frequent use of cellulose-based papers instead of nitrocellulose in LFA devices and thus potentially improve the sustainability in the field of POC diagnostics.


Asunto(s)
Anticuerpos Antivirales/análisis , Prueba Serológica para COVID-19/métodos , COVID-19/diagnóstico , Carbohidratos/química , Colodión/química , Inmunoensayo/métodos , Materiales Biocompatibles , Gonadotropina Coriónica/química , Clostridium thermocellum/inmunología , Humanos , Fragmentos de Inmunoglobulinas/química , Inmunoglobulina G/química , Sistemas de Atención de Punto , Unión Proteica , SARS-CoV-2/inmunología , Urinálisis
15.
Anal Bioanal Chem ; 413(7): 1929-1939, 2021 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-33501551

RESUMEN

The immobilization of sensing bioreceptors is a critical feature affecting the final performance of a biosensor. For DNA detection, the (strept)avidin-biotin affinity interaction is often used for the immobilization of biotin-labeled oligonucleotides or PCR amplicons. Herein, DNA binding proteins are proposed as alternative universal anchors for both DNA immobilization and detection, based on the strong and specific affinity interaction between certain DNA binding proteins and their respective dsDNA binding sites. These binding sites can be incorporated in the target DNA molecule during synthesis and by PCR, eliminating the need for post-synthesis chemical modification and resulting in lower costs. When scCro DNA binding protein was immobilized on microplates and nitrocellulose membrane, both ssDNA and dsDNA targets were successfully detected. The detection limits achieved were similar to those obtained with the streptavidin-biotin system. However, the scCro system resulted in higher signals while using less amount of protein. The adsorption properties of scCro were superior to streptavidin's, making scCro a viable alternative as an anchor biomolecule for the development of DNA assays and biosensors. Finally, a nucleic acid lateral flow assay based solely on two different DNA binding proteins, scCro and dHP, was developed for the detection of a PCR amplicon. Overall, the proposed system appears to be very promising and with potential use for multiplex detection using various DNA binding proteins with different sequence specificities. Further work is required to better understand the adsorption properties of these biomolecules on nitrocellulose, optimize the assays comprehensively, and achieve improved sensitivities.


Asunto(s)
Colodión/análisis , Proteínas de Unión al ADN/química , Ácidos Nucleicos Inmovilizados/química , Adsorción , Sitios de Unión , Técnicas Biosensibles , Biotecnología , Biotina/química , Colodión/química , ADN/química , ADN de Cadena Simple/análisis , Detergentes/química , Límite de Detección , Membranas Artificiales , Reacción en Cadena de la Polimerasa , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Estreptavidina/química
16.
J Biomed Mater Res B Appl Biomater ; 109(10): 1455-1464, 2021 10.
Artículo en Inglés | MEDLINE | ID: mdl-33501724

RESUMEN

This study aimed to develop a sensitive lateral flow test strip for the detection of bisphenol A (BPA) in breast milk. Conventional nitrocellulose test membrane was coated with the coaxial nanofiber, consisting of the inner polycaprolactone (PCL) and the outer PCL/silk fibroin (SF) mixture, to decrease the flow rate of the breast milk in the lateral flow assay (LFA). The nanofiber was prepared by using coaxial electrospinning, and BPA antibody was immobilized physically to the nanofiber. This nanofiber was used as a test membrane in the LFA. Color changes on the test membrane were evaluated as the signal intensity of the BPA. Breast milk creates a background on surfaces due to its structural properties. This background was detected by comparing the signal intensity with the signal intensity of water. The higher signal intensity was found in water samples when compared to breast milk samples. Although the detection limit is 2 ng/ml in both coaxial PCL/SF nanofiber and nitrocellulose (NC) test membranes, the color intensity increased with the increasing BPA concentration in the coaxial PCL/SF nanofiber. As a new dimension, the coaxial PCL/SF nanofiber provided higher color intensity than the NC membrane. In conclusion, a sensitive onsite method was developed for the detection of BPA in breast milk by using new coaxial PCL/SF nanofiber as a test membrane in LFA.


Asunto(s)
Compuestos de Bencidrilo/análisis , Fibroínas/química , Leche Humana/química , Nanofibras/química , Fenoles/análisis , Poliésteres/química , Anticuerpos/química , Anticuerpos/inmunología , Compuestos de Bencidrilo/inmunología , Colodión/química , Femenino , Oro/química , Humanos , Límite de Detección , Nanopartículas del Metal/química , Fenoles/inmunología , Propiedades de Superficie
17.
J Biomater Appl ; 35(6): 592-601, 2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-32873118

RESUMEN

Amniotic membrane (AM) has been shown to enhance corneal wound healing due to the abundance of growth factors, cytokines, and extracellular matrix (ECM) proteins inherent to the tissue. As such, AM has garnered widespread clinical utility as a biological dressing for a number of ophthalmic and soft tissue applications. The preparation, sterilization, and storage procedures used to manufacture AM grafts are extremely important for the conservation of inherent biological components within the membrane. Current processing techniques use harsh chemicals and sterilization agents that can compromise the fundamental wound healing properties of AM. Furthermore, commercially available cryopreserved AM products require specific storage conditions (e.g., ultra-low freezers) thereby limiting their clinical availability in austere environments. Supercritical carbon dioxide (SCCO2) technology allows for the sterilization of biological tissues without the resulting degradation of integral ECM proteins and other factors often seen with current tissue sterilization processes. With this study we demonstrate that lyophilized AM, sterilized using SCCO2, maintains similar biochemical properties and biocompatibility as that of commercially available AM products requiring specialized cold storage conditions.


Asunto(s)
Aloinjertos/química , Amnios/química , Materiales Biocompatibles/química , Dióxido de Carbono/química , Liofilización/métodos , Aloinjertos/metabolismo , Amnios/metabolismo , Animales , Vendajes , Materiales Biocompatibles/metabolismo , Colodión/química , Córnea/metabolismo , Citocinas/metabolismo , Matriz Extracelular/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Masculino , Conejos , Esterilización , Cicatrización de Heridas/efectos de los fármacos
18.
Biotechnol Appl Biochem ; 68(5): 1095-1106, 2021 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-32935878

RESUMEN

Salmonella and Shigella genera are common pathogens that contaminate foods and beverages. Lateral flow assays (LFA) are commonly used to detect these pathogens. However, most of the developed LFAs are for single detection. Simultaneous detection of pathogens is required to reduce cost and time. In this work, 40 nm gold nanoparticles (AuNPs) were synthesized using the seeding growth method as labeling agent. The AuNPs were characterized and conjugated with mouse anti-Gram negative endotoxin antibody. The nitrocellulose membrane HF135 was immobilized with anti-mouse IgG antibody as a control line and two separate test lines with either anti-Shigella or anti-Salmonella antibody, respectively. Color intensity of test lines was observed for positive samples. A milk sample was used as proof of concept to mimic actual contamination. The limit of detection of the LFA was 3.0 × 106 CFU/mL for multiplex detection of Shigella flexneri and Salmonella Typhi and for both single detections. The result was comparable with the enzyme-linked immunosorbent assay (ELISA) analysis. The produced LFA could differentiate between Shigella flexneri, Shigella boydii, Salmonella Enteritidis, and Salmonella Typhi. The developed LFA was able to identify Shigella flexneri and Salmonella Typhi with good sensitivity in milk samples, thus, beneficial to ensure the safety of food before entering the market.


Asunto(s)
Oro/química , Nanopartículas del Metal/química , Técnicas Analíticas Microfluídicas , Salmonella/aislamiento & purificación , Shigella/aislamiento & purificación , Anticuerpos/química , Colodión/química , Ensayo de Inmunoadsorción Enzimática/instrumentación , Diseño de Equipo , Técnicas Analíticas Microfluídicas/instrumentación , Tamaño de la Partícula
19.
Protein Expr Purif ; 177: 105764, 2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-32971296

RESUMEN

Immobilizing antibodies on the nitrocellulose membrane is an important step to increase the sensitivity of the Lateral Flow Test strip for detecting pathogenic antigen. In our research, the fusion protein between nitrocellulose-binding anchor protein 3-Helix - a protein that has a strong affinity to nitrocellulose membrane and protein A - a protein that can bind to the Fc tail of IgG antibody was generated. This fusion protein was expected to help IgG antibodies to be more strongly binding and oriented immobilized onto the nitrocellulose membrane. The recombinant vector pET22b-proA and pET22b-proA-3-Helix coded for protein A and protein A-3-Helix were cloned. These proteins were overexpressed in BL21 and purified by immobilized metal affinity chromatography with purity above 90%. The purified protein was used to evaluate the orientation binding on nitrocellulose membranes by lateral flow challenge. Results showed that protein A-3-Helix binding to nitrocellulose membrane was better than that of protein A. The former protein increased antibody binding and stereochemical immobilizing onto nitrocellulose membrane compared to its protein A counterpart. In summary, we have succeeded in cloning, purifying, and characterizing a dual-head recombinant protein A and protein A-3-Helix. The results show the potential application of protein A-3-Helix in the immobilizing antibody on the test strip.


Asunto(s)
Cromatografía de Afinidad/métodos , Colodión/química , Proteínas Inmovilizadas/química , Fragmentos Fc de Inmunoglobulinas/química , Inmunoglobulina G/química , Proteína Estafilocócica A/química , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Proteínas Inmovilizadas/genética , Proteínas Inmovilizadas/inmunología , Fragmentos Fc de Inmunoglobulinas/genética , Fragmentos Fc de Inmunoglobulinas/inmunología , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Unión Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteína Estafilocócica A/genética , Proteína Estafilocócica A/inmunología
20.
Methods Mol Biol ; 2237: 129-139, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33237414

RESUMEN

Membrane arrays are a unique array platform option for the detection of multiple analytes or materials simultaneously. Their naturally absorptive properties and near universal use in various laboratory methods make it an excellent source with which to probe multiple factors simultaneously. Any liquid sample type can be probed, from bacterial strains, tissue lysates, secreted proteins, to DNA aptamers. Below, we will describe some considerations in how to print a membrane array and then a specific usage of the membrane arrays as it relates to a sandwich-based antibody array technique for simultaneously detection of secreted proteins in a liquid sample.


Asunto(s)
Immunoblotting/métodos , Membranas Artificiales , Análisis por Matrices de Proteínas/métodos , Absorción Fisicoquímica , Colodión/química , Polivinilos/química
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