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1.
J Immunoassay Immunochem ; 45(4): 307-324, 2024 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-38776466

RESUMO

Single Chain Variable Fragment (scFv), a small fragment of antibody can be used to substitute the monoclonal antibody for diagnostic purposes. Production of scFv in Escherichia coli host has been a challenge due to the potential miss-folding and formation of inclusion bodies. This study aimed to express anti-CHIKV E2 scFv which previously designed specifically for Asian strains by co-expression of three chaperones that play a role in increasing protein solubility; GroEL, GroES, and Trigger Factor. The scFv and chaperones were expressed in Origami B E. coli host under the control of the T7 promoter, and purified using a Ni-NTA column. Functional assay of anti-CHIKV-E2 scFv was examined by electrochemical immunosensor using gold modified Screen Printed Carbon Electrode (SPCE), and characterized by differential pulses voltammetry (DPV) using K3[Fe(CN)6] redox system and scanning microscope electron (SEM). The experimental condition was optimized using the Box-Behnken design. The results showed that co-expression of chaperone increased the soluble scFv yield from 54.405 µg/mL to 220.097 µg/mL (~5×). Furthermore, scFv can be used to detect CHIKV-E2 in immunosensor electrochemistry with a detection limit of 0.74048 ng/mL and a quantification limit of 2,24388 ng/mL. Thus, the scFv-anti-CHIKV-E2 can be applied as a bioreceptor in another immunoassay method.


Assuntos
Técnicas Biossensoriais , Técnicas Eletroquímicas , Escherichia coli , Chaperonas Moleculares , Anticorpos de Cadeia Única , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/genética , Escherichia coli/metabolismo , Escherichia coli/genética , Chaperonas Moleculares/imunologia , Imunoensaio/métodos
2.
ACS Appl Bio Mater ; 7(4): 2488-2498, 2024 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-38577953

RESUMO

Green synthesis approaches for making nanosized ceria using starch from cassava as template molecules to control the particle size are reported. The results of the green synthesis of ceria with an optimum calcination temperature of 800 °C shows a size distribution of each particle of less than 30 nm with an average size of 9.68 nm, while the ratio of Ce3+ to Ce4+ was 25.6%. The green-synthesized nanoceria are applied to increase the sensitivity and attach biomolecules to the electrode surface of the electrochemical aptasensor system for coronavirus disease (COVID-19). The response of the aptasensor to the receptor binding domain of the virus was determined with the potassium ferricyanide redox system. The screen-printed carbon electrode that has been modified with green-synthesized nanoceria shows 1.43 times higher conductivity than the bare electrode, while those modified with commercial ceria increase only 1.18 times. Using an optimized parameter for preparing the aptasensors, the detection and quantification limits were 1.94 and 5.87 ng·mL-1, and the accuracy and precision values were 98.5 and 89.1%. These results show that green-synthesized ceria could be a promising approach for fabricating an electrochemical aptasensor.


Assuntos
Técnicas Biossensoriais , COVID-19 , Cério , Manihot , Nanopartículas , Carbono/química , SARS-CoV-2 , Técnicas Eletroquímicas/métodos , Técnicas Biossensoriais/métodos , COVID-19/diagnóstico , Nanopartículas/química , Eletrodos
3.
ACS Appl Bio Mater ; 7(2): 950-960, 2024 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-38303668

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), or coronavirus disease 2019 (COVID-19), is still spreading worldwide; therefore, the need for rapid and accurate detection methods remains relevant to maintain the spread of this infectious disease. Electrochemical immunosensors are an alternative method for the rapid detection of the SARS-CoV-2 virus. Herein, we report the development of a screen-printed carbon electrode immunosensor using a hydroxyapatite-gold nanocomposite (SPCE/HA-Au) directly spray-coated with the immobilization receptor binding domain (RBD) Spike to increase the conductivity and surface electrode area. The HA-Au composite synthesis was optimized using the Box-Behnken method, and the resulting composite was characterized by UV-vis spectrophotometry, TEM-EDX, and XRD analysis. The specific interaction of RBD Spike with immunoglobulin G (IgG) antibodies was evaluated by differential pulse voltammetry and electrochemical impedance spectroscopy methods in a [Fe(CN)6]4-/3- solution redox system. The IgG was detected with a detection limit of 0.0561 pg mL-1, and the immunosensor had selectivity and stability of 103-122% and was stable until week 7 with the influence of storage conditions. Also, the immunosensor was tested using real samples from human serum, where the results were confirmed using the chemiluminescent microparticle immunoassay (CMIA) method and showed satisfactory results. Therefore, the developed electrochemical immunosensor can rapidly and accurately detect SARS-CoV-2 antibodies.


Assuntos
Técnicas Biossensoriais , COVID-19 , Humanos , Carbono/química , Ouro/química , SARS-CoV-2 , Técnicas Biossensoriais/métodos , COVID-19/diagnóstico , Imunoensaio/métodos , Anticorpos Antivirais , Imunoglobulina G , Eletrodos , Hidroxiapatitas
4.
Biomolecules ; 13(12)2023 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-38136624

RESUMO

Fatty-acid-binding proteins (FABPs) serve a crucial role in the metabolism and transport of fatty acids and other hydrophobic ligands as an intracellular protein family. They are also recognized as a critical mediator in the inflammatory and ischemic pathways. FABPs are found in a wide range of tissues and organs, allowing them to contribute to various disease/injury developments that have not been widely discussed. We have collected and analyzed research journals that have investigated the role of FABPs in various diseases. Through this review, we discuss the findings on the potential of FABPs as biomarkers for various diseases in different tissues and organs, looking at their expression levels and their roles in related diseases according to available literature data. FABPs have been reported to show significantly increased expression levels in various tissues and organs associated with metabolic and inflammatory diseases. Therefore, FABPs are a promising novel biomarker that needs further development to optimize disease diagnosis and prognosis methods along with previously discovered markers.


Assuntos
Proteínas de Ligação a Ácido Graxo , Ácidos Graxos , Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Ligação a Ácido Graxo/metabolismo , Ácidos Graxos/metabolismo , Proteínas de Membrana Transportadoras , Biomarcadores
5.
Biosensors (Basel) ; 13(6)2023 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-37367022

RESUMO

Fast, sensitive, and easy-to-use methods for detecting DNA related to food adulteration, health, religious, and commercial purposes are evolving. In this research, a label-free electrochemical DNA biosensor method was developed for the detection of pork in processed meat samples. Gold electrodeposited screen-printed carbon electrodes (SPCEs) were used and characterized using SEM and cyclic voltammetry. A biotinylated probe DNA sequence of the Cyt b S. scrofa gene mtDNA used as a sensing element containing guanine substituted by inosine bases. The detection of probe-target DNA hybridization on the streptavidin-modified gold SPCE surface was carried out by the peak guanine oxidation of the target using differential pulse voltammetry (DPV). The optimum experimental conditions of data processing using the Box-Behnken design were obtained after 90 min of streptavidin incubation time, at the DNA probe concentration of 1.0 µg/mL, and after 5 min of probe-target DNA hybridization. The detection limit was 0.135 µg/mL, with a linearity range of 0.5-1.5 µg/mL. The resulting current response indicated that this detection method was selective against 5% pork DNA in a mixture of meat samples. This electrochemical biosensor method can be developed into a portable point-of-care detection method for the presence of pork or food adulterations.


Assuntos
Técnicas Biossensoriais , DNA Mitocondrial , Animais , Suínos , Estreptavidina , Técnicas Eletroquímicas/métodos , Contaminação de Alimentos , Sondas de DNA , Técnicas Biossensoriais/métodos , Ouro/química , Guanina , Sus scrofa , Eletrodos
6.
RSC Adv ; 13(9): 5874-5884, 2023 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-36816083

RESUMO

A detection method based on an electrochemical aptasensor has been developed as an alternative fast, portable, simple, inexpensive, and high-accuracy detection method for detecting the SARS-CoV-2 Spike Receptor Binding Domain (spike RBD). The CeO2@NH2 functionalized Screen Printed Carbon Electrode (SPCE) was used to immobilize an aminated aptamer of spike RBD protein via glutaraldehyde as a linker. The aptamer's interaction with the SARS-CoV-2 Spike RBD was measured via the [Fe(CN)6]4-/3- redox system signal. Experimental conditions were optimized using a Box-Behnken experimental design and showed that the optimal conditions of the SARS-CoV-2 aptasensor were 1.5 ng mL-1 of aptamer, immobilization of aptamer for 60 minutes, and Spike RBD incubation for 10 minutes. The developed aptasensor was able to detect the standard SARS-CoV-2 Spike RBD with a detection limit of 0.017 ng mL-1 in the range of 0.001-100 ng mL-1. This aptasensor was used to detect salivary and oropharyngeal swab samples of normal individuals with the addition of Spike RBD, and the recoveries were 92.96% and 96.52%, respectively. The testing on nasopharyngeal swab samples of COVID-19 patients showed that the aptasensor results were comparable with the qRT-PCR results. Thus, the developed aptasensor has the potential to be applied as a SARS-CoV-2 rapid test method for clinical samples.

8.
Trop Med Infect Dis ; 7(10)2022 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-36288050

RESUMO

Tropical diseases (TDs) are among the leading cause of mortality and fatality globally. The emergence and reemergence of TDs continue to challenge healthcare system. Several tropical diseases such as yellow fever, tuberculosis, cholera, Ebola, HIV, rotavirus, dengue, and malaria outbreaks have led to endemics and epidemics around the world, resulting in millions of deaths. The increase in climate change, migration and urbanization, overcrowding, and other factors continue to increase the spread of TDs. More cases of TDs are recorded as a result of substandard health care systems and lack of access to clean water and food. Early diagnosis of these diseases is crucial for treatment and control. Despite the advancement and development of numerous diagnosis assays, the healthcare system is still hindered by many challenges which include low sensitivity, specificity, the need of trained pathologists, the use of chemicals and a lack of point of care (POC) diagnostic. In order to address these issues, scientists have adopted the use of CRISPR/Cas systems which are gene editing technologies that mimic bacterial immune pathways. Recent advances in CRISPR-based biotechnology have significantly expanded the development of biomolecular sensors for diagnosing diseases and understanding cellular signaling pathways. The CRISPR/Cas strategy plays an excellent role in the field of biosensors. The latest developments are evolving with the specific use of CRISPR, which aims for a fast and accurate sensor system. Thus, the aim of this review is to provide concise knowledge on TDs associated with mosquitoes in terms of pathology and epidemiology as well as background knowledge on CRISPR in prokaryotes and eukaryotes. Moreover, the study overviews the application of the CRISPR/Cas system for detection of TDs associated with mosquitoes.

9.
Anal Biochem ; 658: 114902, 2022 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-36122603

RESUMO

The development of the Coronavirus disease 2019 (COVID-19) vaccine is one of the most important efforts in controlling the pandemic. Serological tests are used to identify highly reactive human donors for convalescent plasma therapy, measuring vaccine efficacy and durability. This review article presents a review of serology tests and how antibody titers in response to vaccines have been developed. Some of the serological test methods discussed are Plaque Reduction Neutralization Test (PRNT), Enzyme-Linked Immunosorbent Assay (ELISA), Lateral flow immunoassay (LFIA), chemiluminescent immunoassay (CLIA), and Chemiluminescent Micro-particle Immunoassay (CMIA). This review can provide an understanding of the application of the body's immune response to vaccines to get some new strategies for vaccines.


Assuntos
COVID-19 , SARS-CoV-2 , Humanos , COVID-19/diagnóstico , COVID-19/prevenção & controle , Técnicas de Laboratório Clínico/métodos , Anticorpos Antivirais , Testes Sorológicos/métodos , Ensaio de Imunoadsorção Enzimática/métodos , Vacinação , Anticorpos Neutralizantes , Soroterapia para COVID-19
10.
Adv Pharmacol Pharm Sci ; 2022: 1677621, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35873075

RESUMO

WHO declared the outbreak of COVID-19, caused by SARS-CoV-2, a pandemic in March 2020. More than 223 million cases and approximately 4.6 million deaths have been confirmed. Early diagnosis and immediate treatment became a priority during this pandemic. However, COVID-19 diagnostic testing resources are limited, especially early in the pandemic. Apart from being limited, the COVID-19 diagnostic tests using reverse transcription polymerase chain reaction (RT-PCR) have encountered storage, transportation, and safety issues. These problems are mainly experienced by developing poor countries, countries in the equatorial region, and archipelagic countries. VITPAD® is a guanidine-based inactivation transport medium (ITM) formulated to maintain the RNA quality of SARS-CoV-2 during transportation without cold chains. This study, conducted from September 2020 to March 2021, performed clinical validation of VITPAD® by comparing its performance with a globally commercially available ITM from the NEST brand. Its stability at room temperature, safety, and resistance at high temperatures was also tested using RT-PCR analysis. VITPAD® can reduce the infectious nature of the specimen, preserve the SARS-CoV-2 for 18 days at an ambient temperature, and resist high temperatures (40°C for 3 hours). A guanidine-based transport medium, such as VITPAD®, is compatible and recommended for RT-PCR-based molecular diagnosis of COVID-19.

11.
R Soc Open Sci ; 8(2): 202040, 2021 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-33972878

RESUMO

Epithelial sodium channel (ENaC) is a transmembrane protein that has an essential role in maintaining the levels of sodium in blood plasma. A person with a family history of hypertension has a high enough amount of ENaC protein in the kidneys or other organs, so that the ENaC protein acts as a marker that a person is susceptible to hypertension. An aptasensor involves aptamers, which are oligonucleotides that function similar to antibodies, as sensing elements. An electrochemical aptasensor for the detection of ENaC was developed using a screen-printed carbon electrode (SPCE) which was modified by electrodeposition of cerium oxide (CeO2). The aptamer immobilization was via the streptavidin-biotin system. The measurement of changes in current of the active redox [Fe(CN)6]3-/4- was carried out by differential pulse voltammetry. The surfaces of SPCE and SPCE/CeO2 were characterized using scanning electron microscopy, voltammetry and electrochemical impedance spectroscopy. The Box-Behnken experimental optimization design revealed the streptavidin incubation time, aptamer incubation time and streptavidin concentrations were 30 min, 30 min and 10.8 µg ml-1, respectively. Various concentrations of ENaC were used to obtain the linearity range of 0.05-3.0 ng ml-1, and the limits of detection and quantification were 0.012 ng ml-1 and 0.038 ng ml-1, respectively. This aptasensor method has the potential to measure the ENaC protein levels in urine samples as well as to be a point-of-care device.

12.
RSC Adv ; 11(31): 18737, 2021 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-35481963

RESUMO

[This corrects the article DOI: 10.1039/D1RA00637A.].

13.
RSC Adv ; 11(27): 16216-16235, 2021 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-35479153

RESUMO

Nanoceria (cerium oxide nanoparticles: CeO2-NPs) has received significant attention due to its biocompatibility, good conductivity, and the ability to transfer oxygen. Nanoceria has been widely used to develop electrochemical sensors and biosensors as it could increase response time, sensitivity, and stability of the sensor. In this review, we discussed synthesis methods, and the recent applications employing CeO2-NPs for electrochemical detection of various analytes reported in the most recent four years.

14.
Appl Biochem Biotechnol ; 170(1): 44-57, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23468006

RESUMO

α-Amylase catalyzes hydrolysis of starch to oligosaccharides, which are further degraded to simple sugars. The enzyme has been widely used in food and textile industries and recently, in generation of renewable energy. An α-amylase from yeast Saccharomycopsis fibuligera R64 (Sfamy) is active at 50 °C and capable of degrading raw starch, making it attractive for the aforementioned applications. To improve its characteristics as well as to provide information for structural study ab initio, the enzyme was chemically modified by acid anhydrides (nonpolar groups), glyoxylic acid (GA) (polar group), dimethyl adipimidate (DMA) (cross-linking), and polyethylene glycol (PEG) (hydrophilization). Introduction of nonpolar groups increased enzyme stability up to 18 times, while modification by a cross-linking agent resulted in protection of the calcium ion, which is essential for enzyme activity and integrity. The hydrophilization with PEG resulted in protection against tryptic digestion. The chemical modification of Sfamy by various modifiers has thereby resulted in improvement of its characteristics and provided systematic information beneficial for structural study of the enzyme. An in silico structural study of the enzyme improved the interpretation of the results.


Assuntos
Proteínas Fúngicas/química , Engenharia de Proteínas/métodos , Saccharomycopsis/química , alfa-Amilases/química , Anidridos Acéticos/química , Sequência de Aminoácidos , Quelantes/química , Reagentes de Ligações Cruzadas/química , Dimetil Adipimidato/química , Estabilidade Enzimática , Glioxilatos/química , Temperatura Alta , Hidrólise , Modelos Moleculares , Dados de Sequência Molecular , Polietilenoglicóis/química , Proteólise , Saccharomycopsis/enzimologia , Amido/metabolismo
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