Development of a new accurate lateral flow immunoassay for diagnosis of human leptospirosis.

PURPOSE: The current diagnostic methods for leptospirosis diagnosis are technically complex and expensive, with limited applicability to specialized laboratories. Furthermore, they lack diagnostic accuracy in the acute stage of the disease, which coincides with a period when antibiotics are highly effective. New simple and accurate tests are mandatory to decentralize and improve diagnosis. Here, we introduced a new lateral flow immunoassay (Lepto-LF) for human leptospirosis. METHODS: We conducted a double-blinded assay using 104 serum samples from patients with confirmed or discarded diagnosis for leptospirosis. The diagnostic performance of Lepto-LF was estimated across different ranges of days from onset of symptoms (dpo), considering the diagnostic algorithm as reference standard. Additionally, it was compared with the screening methods enzyme-linked immunosorbent assay (IgM-ELISA) and the slide agglutination test using temperature-resistant antigen (SATR). RESULTS: Lepto-LF exhibited perfect diagnostic performance with a Youden´s index J = 1 from 6 dpo in the acute phase. IgM-ELISA gave slightly lower accuracy with J = 0.91 and 95.5% of both sensitivity and specificity; while SATR showed a markedly inferior yield (J = 0.41, sensitivity = 95.5%, specificity = 45.5%). The performances remained consistent in the convalescence phase of the disease (> 10 dpo). CONCLUSION: Lepto-LF was found to be a reliable test for simple, rapid and early diagnosis of leptospirosis, resulting a promising tool for decentralizing leptospirosis diagnosis and enabling timely treatment of patients. In addition, Lepto-LF may be employed as confirmatory test, especially in remote areas and vulnerable contexts where the standard MAT is not available.

Evaluation of concordance of new QuantiFERON-TB Gold Plus platforms for Mycobacterium tuberculosis infection diagnosis in a prospective cohort of household contacts.

Interferon-gamma (IFN-γ) release assays play a pivotal role in tuberculosis infection (TBI) diagnosis, with QuantiFERON-TB Gold Plus-an enzyme-linked immunosorbent assay (ELISA)-among the most widely utilized. Newer QuantiFERON-TB platforms with shorter turnaround times were recently released. We aimed to evaluate these platforms' agreement in the diagnosis of TBI. Blood samples from a prospective cohort of tuberculosis household contacts were collected at baseline and after 12 weeks of follow-up, and tested with LIAISON, an automated chemiluminescence immunoassay (CLIA) system, QIAreach, a lateral flow (QFT-LF) semi-automated immunoassay, and the ELISA QuantiFERON-TB Gold Plus platform. Test concordances were analyzed. ELISA vs CLIA overall agreement was 83.3% for all tested samples (120/144) [Cohen's kappa coefficient (κ): 0.66 (95% CI: 0.54-0.77)]. Samples positive with CLIA provided consistently higher IFN-γ levels than with ELISA (P < 0.001). Twenty-four (16.7%) discordant pairs were obtained, all CLIA-positive/ELISA-negative: 15 (62.5%) had CLIA IFN-γ levels within borderline values (0.35-0.99 IU/mL) and 9 (37.5%) >0.99 IU/mL. QFT-LF showed only 76.4% (68/89) overall agreement with ELISA [κ: 0.53 (95% CI: 0.37-0.68)] with 21 (23.6%) discordant results obtained, all QFT-LF-positive/ELISA-negative. Overall concordance between ELISA and CLIA platforms was substantial, and only moderate between ELISA and QFT-LF. The CLIA platform yielded higher IFN-γ levels than ELISA, leading to an almost 17% higher positivity rate. The techniques do not seem interchangeable, and validation against other gold standards, such as microbiologically-confirmed tuberculosis disease, is required to determine whether these cases represent true new infections or whether CLIA necessitates a higher cutoff. IMPORTANCE: Tuberculosis is an airborne infectious disease caused by Mycobacterium tuberculosis that affects over 10 million people annually, with over 2 billion people carrying an asymptomatic tuberculosis infection (TBI) worldwide. Currently, TBI diagnosis includes tuberculin skin test and the blood-based interferon-gamma (IFN-γ) release assays, with Qiagen QuantiFERON-TB Gold Plus (QFT) being among those most widely utilized. We evaluated Qiagen's newer QFT platforms commercially available in a prospective cohort of tuberculosis contacts. A substantial agreement was obtained between the current QFT-enzyme-linked immunosorbent assay (ELISA) and the new QFT-chemiluminescence immunoassay (CLIA) platform, although QFT-CLIA provided higher concentrations of IFN-γ, leading to a 16.6% higher positivity rate. We highlight that both platforms may not be directly interchangeable and that further validation is required.

Improving disseminated histoplasmosis diagnosis in HIV/AIDS patients in Suriname: The role of a urine lateral flow assay.

Histoplasmosis is a frequent cause of infections in people living with HIV/AIDS (PLWHA). This study introduces the application of a Histoplasma capsulatum urine antigen lateral flow assay (LFA) for diagnosing disseminated histoplasmosis in PLWHA in Suriname. The LFA's diagnostic accuracy was compared with the current diagnostic approach, aiming to assess whether this test resulted in improved early detection and management. Additionally, the prevalence of histoplasmosis among advanced stage HIV patients without clinical suspicion of infection was evaluated using the same LFA. In total, 98 patients were included in the study, of which 58 were classified as "possible disseminated histoplasmosis (DH)" based on clinical criteria and 40 as "controls". Of these possible DH cases, only 19 (32.7%) had a positive LFA. During the study, decisions for treatment were made without the treating physician being aware of the LFA result. Only 55% of the patients who started treatment for histoplasmosis based on clinical criteria had a positive LFA, and 21% of untreated patients had a positive LFA. This study shows that combining clinical signs with LFA results enhances diagnostic accuracy and is cost effective, resulting in better treatment decisions.

Utilizing COVID-19 as a Model for Diagnostics Using an Electrochemical Sensor.

This paper reports a rapid and sensitive sensor for the detection and quantification of the COVID-19 N-protein (N-PROT) via an electrochemical mechanism. Single-frequency electrochemical impedance spectroscopy was used as a transduction method for real-time measurement of the N-PROT in an immunosensor system based on gold-conjugate-modified carbon screen-printed electrodes (Cov-Ag-SPE). The system presents high selectivity attained through an optimal stimulation signal composed of a 0.0 V DC potential and 10 mV RMS-1 AC signal at 100 Hz over 300 s. The Cov-Ag-SPE showed a log response toward N-PROT detection at concentrations from 1.0 ng mL-1 to 10.0 µg mL-1, with a 0.977 correlation coefficient for the phase (θ) variation. An ML-based approach could be created using some aspects observed from the positive and negative samples; hence, it was possible to classify 252 samples, reaching 83.0, 96.2 and 91.3% sensitivity, specificity, and accuracy, respectively, with confidence intervals (CI) ranging from 73.0 to 100.0%. Because impedance spectroscopy measurements can be performed with low-cost portable instruments, the immunosensor proposed here can be applied in point-of-care diagnostics for mass testing, even in places with limited resources, as an alternative to the common diagnostics methods.

Development of optical and electrochemical immunodevices for dengue virus detection.

Dengue virus (DENV) is the most prevalent global arbovirus, exhibiting a high worldwide incidence with intensified severity of symptoms and alarming mortality rates. Faced with the limitations of diagnostic methods, an optical and electrochemical biosystem was developed for the detection of DENV genotypes 1 and 2, using cysteine (Cys), cadmium telluride (CdTe) quantum dots, and anti-DENV antibodies. Cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), surface plasmon resonance (SPR), atomic force microscopy (AFM), and Fourier transform infrared spectroscopy (FTIR) were employed to characterize the immunosensor. The AFM and SPR results demonstrated discernible topographic and angular changes confirming the biomolecular recognition. Different concentrations of DENV-1 and DENV-2 were evaluated (0.05 × 106 to 2.0 × 106 PFU mL-1), resulting in a maximum anodic shift (ΔI%) of 263.67% ± 12.54 for DENV-1 and 63.36% ± 3.68 for DENV-2. The detection strategies exhibited a linear response to the increase in viral concentration. Excellent linear correlations, with R2 values of 0.95391 for DENV-1 and 0.97773 for DENV-2, were obtained across a broad concentration range. Data analysis demonstrated high reproducibility, displaying relative standard deviation values of 3.42% and 3.62% for Cys-CdTe-antibodyDENV-1-BSA and Cys-CdTe-antibodyDENV-2-BSA systems. The detection limits were 0.34 × 106 PFU mL-1 and 0.02 × 106 PFU mL-1, while the quantification limits were set at 1.49 × 106 PFU mL-1 and 0.06 × 106 PFU mL-1 for DENV-1 and DENV-2, respectively. Therefore, the biosensing apparatus demonstrates analytical effectiveness in viral screening and can be considered an innovative solution for early dengue diagnosis, contributing to global public health.

Point-of-Care Electrochemical Immunosensor Applied against Nosocomial Infection: Staphylococcus aureus Detection in Human Hand Skin.

Staphylococcus aureus is an important pathogen that causes nosocomial infections, resulting in unacceptable morbidity and mortality rates. In this work, we proposed the construction of a nanostructured ZnO-based electrochemical immunosensor for qualitative and semiquantitative detection of S. aureus using simple methods for growing zinc oxide nanorods (ZnO NRs) on a sensor board and immobilizing the anti-S. aureus antibody on ZnO NRs through cystamine and glutaraldehyde. The immunosensor detected S. aureus in the 103-107 colony-forming unit (CFU) mL-1 range and showed a limit of detection (LoD) around 0.792 × 103 CFU mL-1. Beyond a satisfactory LoD, the developed immunosensor presented other advantages, such as high versatility for point-of-care assays and a suitable selective factor that admits the detection of the S. aureus concentration range in human hand skin after washing. Moreover, the immunosensor showed the potential to be an excellent device to control nosocomial infection by detecting the presence of S. aureus in human hand skin.

Electrochemical microfluidic immunosensor with graphene-decorated gold nanoporous for T-2 mycotoxin detection.

T-2 is one of the most potent cytotoxic food-borne mycotoxins. In this work, we have developed and characterized an electrochemical microfluidic immunosensor for T-2 toxin quantification in wheat germ samples. T-2 toxin detection was carried out using a competitive immunoassay method based on monoclonal anti-T-2 antibodies immobilized on the poly(methyl methacrylate) (PMMA) microfluidic central channel. The platinum wire working electrode at the end of the channel was in situ modified by a single-step electrodeposition procedure with reduced graphene oxide (rGO)-nanoporous gold (NPG). T-2 toxin in the sample was allowed to compete with T-2-horseradish peroxidase (HRP) conjugated for the specific recognizing sites of immobilized anti-T-2 monoclonal antibodies. The HRP, in the presence of hydrogen peroxide (H2O2), catalyzes the oxidation of 4-tert-butylcatechol (4-TBC), whose back electrochemical reduction was detected on the nanostructured electrode at -0.15 V. Thus, at low T-2 concentrations in the sample, more enzymatically conjugated T-2 will bind to the capture antibodies, and, therefore, a higher current is expected. The detection limits found for electrochemical immunosensor, and commercial ELISA procedure were 0.10 µg kg-1 and 10 µg kg-1, and the intra- and inter-assay coefficients of variation were below 5.35% and 6.87%, respectively. Finally, our microfluidic immunosensor to T-2 toxin will significantly contribute to faster, direct, and secure in situ analysis in agricultural samples.

Single-Response Duplexing of Electrochemical Label-Free Biosensor from the Same Tag.

Multiplexing is a valuable strategy to boost throughput and improve clinical accuracy. Exploiting the vertical, meshed design of reproducible and low-cost ultra-dense electrochemical chips, the unprecedented single-response multiplexing of typical label-free biosensors is reported. Using a cheap, handheld one-channel workstation and a single redox probe, that is, ferro/ferricyanide, the recognition events taking place on two spatially resolved locations of the same working electrode can be tracked along a single voltammetry scan by collecting the electrochemical signatures of the probe in relation to different quasi-reference electrodes, Au (0 V) and Ag/AgCl ink (+0.2 V). This spatial isolation prevents crosstalk between the redox tags and interferences over functionalization and binding steps, representing an advantage over the existing non-spatially resolved single-response multiplex strategies. As proof of concept, peptide-tethered immunosensors are demonstrated to provide the duplex detection of COVID-19 antibodies, thereby doubling the throughput while achieving 100% accuracy in serum samples. The approach is envisioned to enable broad applications in high-throughput and multi-analyte platforms, as it can be tailored to other biosensing devices and formats.

Una plaga citadina peligrosa: los murciélagos / A dangerous urban pest: bats

Los murciélagos son mamíferos vertebrados presentes en la Ciudad de Buenos Aires, estimándose una población de 4 animales por habitante. Son portadores de varias enfermedades importantes y además empeoran las condiciones respiratorias de enfermos crónicos. En el campo cumplen una interesante función, ya que se alimentan de insectos perjudiciales para las siembras. El guano puede ser útil en el abono de la tierra debido al aporte de carbono y nitrógeno. En las ciudades su presencia tiene consecuencias diferentes. Se encuentran en los taparrollos de las habitaciones, así como también en todas las oquedades de muros, árboles, grietas, etc. Se exponen aquí los peligros y los cuidados que deben tenerse en la Ciudad de Buenos Aires ante la invasión de estos quirópteros. (AU)

Bats are vertebrate mammals present in the City of Buenos Aires, with an estimated population of 4 animals per inhabitant. They are carriers of several important diseases and also worsen the respiratory conditions of the chronically ill. In rural areas they fulfill an interesting function, since they feed on insects harmful to crops. Guano can be useful in soil fertilization due to its contribution of carbon and nitrogen. In cities their presence has different consequences. They are found in the roll covers of the rooms as well as in all the hollows of walls, trees, cracks, etc. The dangers and precautions to be taken in the city of Buenos Aires in the face of the invasion of these chiroptera are described here. (AU)

Membraneless, self-powered immunosensing of a cardiac biomarker by exploiting a PEC platform based on CaBi2Ta2O9 combined with bismuth oxyiodides.

This work describes the development of a membraneless, self-powered immunosensor exploiting a photoelectrochemical system based on two photoelectrodes for cardiac troponin I (cTn). An electrode based on CaBi2Ta2O9 combined with bismuth oxyiodides (BiOI/Bi4O5I2/Bi5O7I) was modified with the cTnI antibody (anti-cTnI) and applied in a photoelectrochemical cell as a photoanode. To perform the cTnI detection exploiting a self-powered photoelectrochemical setup, the immunosensor (anti-cTnI/BiOI/Bi4O5I2/Bi5O7I/CaBi2Ta2O9/FTO) was coupled to a photoelectrochemical cell containing a photocathode based on CuBi2O4 (CBO/FTO) for zero-biased photoelectrochemical immunosensing of cardiac troponin I (cTnI) biomarker. For comparison purposes, the photoanode was applied for cTnI detection in a three-electrode electrochemical cell. The spectroscopic, structural, and morphological characteristics of the photoelectrochemical (PEC) materials were evaluated using scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDS), and X-ray diffraction (XRD). Electrochemical impedance spectroscopy (EIS) measurements were performed in the presence and absence of light to investigate the effects of photons on the charge transfer resistance of the photoanode. The influence of the cTnI biomarker on the photoelectrochemical response of the anti-cTnI antibody-modified photoelectrochemical platform (anti-cTnI/BiOI/Bi4O5I2/Bi5O7I/CaBi2Ta2O9/FTO) was evaluated by measuring the photocurrent of the system. The immunosensor presented a linear response ranging from 1 pg mL-1 to 200 ng mL-1 as well as a mean recovery percentage between 95.7% and 108.0% in real human serum samples for the cTnI biomarker.

Actualización de informe de prueba de quimioluminiscencia para diagnóstico de COVID-19: (17 de agosto del 2023) / COVID-19 Diagnostic Chemiluminescence Test Report Update: (August 17, 2023)

ANTECEDENTES: Según la REVISIÓN RÁPIDA N°02-2020 realizado en Agosto del 2020, por solicitud del Equipo Funcional de Infectología, se realiza una revisión rápida con el fin de realizar una búsqueda y análisis de Ia mejor evidencia científica disponible en relación a la utilización de la prueba serológica por quimioluminiscencia para detección de SARS-COV-2. La Unidad Funcional de Evaluación de Tecnologías Sanitarias de INEN se creó el 15 de enero del 2020 mediante R.J. 020-2020-J/INEN y dentro de sus funciones están el "Evaluar aquellas tecnologías sanitarias requeridas por órganos usuarios, que sean nuevas para la entidad y/o no cuenten con cobertura financiera para la/s IAFAS". Definiendo tecnologías sanitarias a "cualquier intervención que pueda ser utilizada en la promoción de la salud, prevención, diagnóstico o tratamiento de una enfermedad, rehabilitación o cuidados prolongados. Se incluyen los medicamentos, los dispositivos, los procedimientos médicos y quirúrgicos, así como los sistemas organizativos dentro de los cuales se proporciona dicha atención sanitaria". Dentro de las funciones de UFETS-INEN es re-evaluar tecnologías que fueron evaluadas previamente con alguna recomendación en contra o una aprobación que requiera evaluación. METODOLOGÍA PARA ACTUALIZACIÓN: En el presente documento se hace una actualización a la metodología usada en el primer informe, actualización de resultados de la búsqueda científica y de estudios. No se cambiará la pregunta PICO. ACERCA DE LA TECNOLOGÍA: La pandemia por el nuevo coronavirus durante el 2019 (COVID-19) originado en Wuhan, China, generó hasta la fecha más de 6 millones de muertes2 en todo el mundo. La mayoría de las personas infectadas con este virus desarrolló una forma leve y el 12%-32% requirió un ingreso a la Unidad de Cuidados Intensivos. La quimioluminiscencia o CLIA es una reacción química que consiste en la emisión de luz por parte de algunas sustancias cuando reaccionan entre sí, con ello se puede realizar inmunoensayos, que son técnicas analíticas que utilizan anticuerpos. Los inmunoensayos CLIA se basan en el mismo principio que los inmunoensayos (ELISA), pero con la diferencia de que el anticuerpo de detección lleva acoplada una enzima que cataliza una reacción quimioluminiscente, produciendo una señal luminosa proporcional a la cantidad de analito presente en la muestra. Para la fecha de la emisión de la evaluación de tecnología sanitaria de quimioluminiscencia para el diagnóstico de SARS-CoV-2 (Agosto 2020), se contaba con el documento normativo con la Resolución Ministerial N* 139-2020-MINSA que aprobó el Documento Técnico: Prevención y Atención de personas afectadas por COVID-19 en el Perú. En este documento se establece la realización de pruebas diagnósticas moleculares y debido a limitaciones de disponibilidad, infraestructura, equipos biomédicos y personal de salud disponible a nivel nacional necesarios para su realización se optó por incorporar pruebas rápidas serológicas como una estrategia de detección de casos en pacientes y profesionales de la salud". En el apartado de tamizaje de dicho documento se establece "En el escenario de transmisión comunitaria, con la finalidad de fortalecer las medidas de contención, es necesario implementar estrategias de tamizaje con la Prueba Rápida IgM/IgG para COVID-19 en personas asintomáticas, pero que se encuentran en mayor riesgo de infección". Se especifica también un instructivo de cómo realizar la toma de muestra y como reportar la información generada. METODOLOGÍA: Primero se realizó una revisión de los documentos que fueron enviados a la unidad y se actualizó la estrategia de búsqueda del Instituto Nacional de Enfermedades Neoplásicas (INEN). ANÁLISIS: La principal evidencia de la revisión rápida anteriormente publicada provino de un estudio de revisión sistemática y meta-análisis de Lisboa et al7 , que evaluaron la exactitud diagnóstica de las pruebas serológicas para COVID-19. Como parte del diseño del estudio se realizó una búsqueda sistemática en Medline, BbioRxiv y medRxiv de enero a abril del presente año siguiendo una estrategia de búsqueda que permitió identificar los principales estudios relacionados a COVID-19 y pruebas serológicas para COVID-19. Se compararon las medidas de sensibilidad o especificidad con un referente estándar por PCR. Los riesgos de sesgos fueron identificados utilizando la herramienta QUADAS-2. Con la búsqueda sistemática se lograron identificar 5016 estudios de los cuales se eligió 40 estudios según criterios de inclusión y exclusión. La sensibilidad tomando en cuenta ambos anticuerpos en las pruebas de ELISA que miden IgG o IgM fue del 84,3% (intervalo de confianza del 95% del 75,6% al 90,9%), de las pruebas LFA fue del 66,0% (49,3% al 79,3%) y de las pruebas serológicas CLIA del 97,8% (46,2% al 100%). La Especificidad tomando en cuenta ambos anticuerpos en las pruebas de ELISA que miden IgG o IgM fue del 97.6% (intervalo de confianza del 95% del 93,2% al 99,4%), de las pruebas LFA fue del 96,6% (94,3% al 98,2%) y de las pruebas serológicas CLIA del 97,8% (62,9% al 99,9%). En todos los análisis, la sensibilidad agrupada de ambos anticuerpos fue menor para los LFA. Se evaluaron 49 riesgos de sesgos encontrándose el 98% de los riesgos en los estudios seguidos de riesgos en la interpretación en el 73%. Para cada uno de los métodos con respecto a sensibilidad y especifica no fueron asociados a las medidas de inmunoglobulinas. Se evidencio sesgos de heterogeneidad en todos los análisis. La sensibilidad fue mayor cuando las pruebas fueron realizadas por lo menos 03 semanas después del inicio de síntomas. Cabe mencionar que este estudio es previo a la era de vacunación de SARS-CoV-2, por lo que todos los estudios no tomaron en cuenta los aspectos relevantes durante la vacunación. CONCLUSIONES: En base a las funciones de UFETS-INEN se actualizó el documento de revisión rápida del uso de pruebas de quimioluminiscencia para el diagnóstico de COVID19. La experiencia del uso de las pruebas CLIA para el diagnóstico de COVID-19 fue necesario e importante durante la era pre-vacunas, sin embargo, después ya no han sido utilizados. En el proceso actual post-pandemia, la vacunación de SARS-CoV-2 ha logrado disminuir los casos severos de COVID-19. Además, durante periodos de prevalencias bajas, el rendimiento de la prueba CLIA puede verse disminuido. Nuestra conclusión es que el uso de la prueba de quimioluminiscencia para antígenos de SARS-CoV-2 durante la era post-vacunación servirá con el fin de revelar los individuos que requieren un refuerzo adicional temprano y/o tardío, para investigación o estudios de seroprevalencias, mas no debe usarse para el diagnóstico de pacientes con COVID-19. Se recomienda enviar a la oficina de seguros este informe para su evaluación.

A highly sensitive nanobody-based immunoassay detecting SARS-CoV-2 nucleocapsid protein using all-recombinant reagents.

Antigen tests have been crucial for managing the COVID-19 pandemic by identifying individuals infected with SARS-CoV-2. This remains true even after immunity has been widely attained through natural infection and vaccination, since it only provides moderate protection against transmission and is highly permeable to the emergence of new virus variants. For this reason, the widespread availability of diagnostic methods is essential for health systems to manage outbreaks effectively. In this work, we generated nanobodies to the virus nucleocapsid protein (NP) and after an affinity-guided selection identified a nanobody pair that allowed the detection of NP at sub-ng/mL levels in a colorimetric two-site ELISA, demonstrating high diagnostic value with clinical samples. We further modified the assay by using a nanobody-NanoLuc luciferase chimeric tracer, resulting in increased sensitivity (detection limit = 61 pg/mL) and remarkable improvement in diagnostic performance. The luminescent assay was finally evaluated using 115 nasopharyngeal swab samples. Receiver Operating Characteristic (ROC) curve analysis revealed a sensitivity of 78.7% (95% confidence interval: 64.3%-89.3%) and specificity of 100.0% (95% confidence interval: 94.7%-100.0%). The test allows the parallel analysis of a large number of untreated samples, and fulfills our goal of producing a recombinant reagent-based test that can be reproduced at low cost by other laboratories with recombinant expression capabilities, aiding to build diagnostic capacity.

Development of a surface plasmon resonance based immunosensor for diclofenac quantification in water.

A Surface Plasmon Resonance (SPR) biosensor based on an inhibition immunoassay was developed for the detection of diclofenac (DCF) in aqueous solution. Due to the small size of DCF, an hapten-protein conjugate was produced by coupling DCF to bovine serum albumin (BSA). DCF-BSA conjugate formation was confirmed via MALDI-TOF mass spectrometry. The resulting conjugate was immobilized onto the surface of a sensor fabricated via e-beam deposition of a 2 nm chromium adhesion layer followed by a 50 nm gold layer onto precleaned BK7 glass slides. Immobilization onto the nano thin gold surface was accomplished by covalent amide linkage through a self-assembled monolayer. Samples were composed of a mixture of antibody at a fixed concentration and DCF at different known concentrations in deionized water, causing the inhibition of anti-DCF on the sensor. The DCF-BSA was obtained with a ratio of 3 DCF molecules per BSA. A calibration curve was performed using concentrations between 2 and 32 µg L-1. The curve was fitted using the Boltzmann equation, reaching a limit of detection (LOD) of 3.15 µg L-1 and limit of quantification (LOQ) of 10.52 µg L-1, the inter-day precision was calculated and an RSD value of 1.96% was obtained; and analysis time of 10 min. The developed biosensor is a preliminary approach to the detection of DCF in environmental water samples, and the first SPR biosensor developed for DCF detection using a hapten-protein conjugate.

Microfluidic Platform Integrated with Carbon Nanofibers-Decorated Gold Nanoporous Sensing Device for Serum PSA Quantification.

Prostate cancer is a disease with a high incidence and mortality rate in men worldwide. Serum prostate-specific antigens (PSA) are the main circulating biomarker for this disease in clinical practices. In this work, we present a portable and reusable microfluidic device for PSA quantification. This device comprises a polymethyl methacrylate microfluidic platform coupled with electrochemical detection. The platinum working microelectrode was positioned in the outflow region of the microchannel and was modified with carbon nanofibers (CNF)-decorated gold nanoporous (GNP) structures by the dynamic hydrogen bubble template method, through the simultaneous electrodeposition of metal precursors in the presence of CNF. CNF/GNP structures exhibit attractive properties, such as a large surface to volume ratio, which increases the antibody's immobilization capacity and the electroactive area. CNFs/GNP structures were characterized by scanning electron microscopy, energy dispersive spectrometry, and cyclic voltammetry. Anti-PSA antibodies and HRP were employed for the immune-electrochemical reaction. The detection limit for the device was 5 pg mL-1, with a linear range from 0.01 to 50 ng mL-1. The coefficients of variation within and between assays were lower than 4.40%, and 6.15%, respectively. Additionally, its clinical performance was tested in serum from 30 prostate cancer patients. This novel device was a sensitive, selective, portable, and reusable tool for the serological diagnosis and monitoring of prostate cancer.

Enhancing a SARS-CoV-2 nucleocapsid antigen test sensitivity with cost efficient strategy through a cotton intermembrane insertion.

Lateral flow antigen tests have been widely used in the Covid-19 pandemic, allowing faster diagnostic test results and preventing further viral spread through isolation of infected individuals. Accomplishment of this screening must be performed with tests that show satisfactory sensitivity in order to successfully detect the target protein and avoid false negatives. The aim of this study was to create a lateral flow test that could detect SARS-CoV-2 nucleocapsid protein in low concentrations that were comparable to the limits of detection claimed by existing tests from the market. To do so, several adjustments were necessary during research and development of the prototypes until they were consistent with these criteria. The proposed alternatives of increasing the test line antibody concentration and addition of an intermembrane between the conjugate pad and the nitrocellulose membrane were able to increase the sensitivity four-fold and generate a new rapid test prototype called "lateral flow intermembrane immunoassay test" (LFIIT). This prototype showed an adequate limit of detection (2.0 ng mL-1) while maintaining affordability and simplicity in manufacturing processes.

Flexible Label-Free Platinum and Bio-PET-Based Immunosensor for the Detection of SARS-CoV-2.

The demand for new devices that enable the detection of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) at a relatively low cost and that are fast and feasible to be used as point-of-care is required overtime on a large scale. In this sense, the use of sustainable materials, for example, the bio-based poly (ethylene terephthalate) (Bio-PET) can be an alternative to current standard diagnostics. In this work, we present a flexible disposable printed electrode based on a platinum thin film on Bio-PET as a substrate for the development of a sensor and immunosensor for the monitoring of COVID-19 biomarkers, by the detection of L-cysteine and the SARS-CoV-2 spike protein, respectively. The electrode was applied in conjunction with 3D printing technology to generate a portable and easy-to-analyze device with a low sample volume. For the L-cysteine determination, chronoamperometry was used, which achieved two linear dynamic ranges (LDR) of 3.98-39.0 µmol L-1 and 39.0-145 µmol L-1, and a limit of detection (LOD) of 0.70 µmol L-1. The detection of the SARS-CoV-2 spike protein was achieved by both square wave voltammetry (SWV) and electrochemical impedance spectroscopy (EIS) by a label-free immunosensor, using potassium ferro-ferricyanide solution as the electrochemical probe. An LDR of 0.70-7.0 and 1.0-30 pmol L-1, with an LOD of 0.70 and 1.0 pmol L-1 were obtained by SWV and EIS, respectively. As a proof of concept, the immunosensor was successfully applied for the detection of the SARS-CoV-2 spike protein in enriched synthetic saliva samples, which demonstrates the potential of using the proposed sensor as an alternative platform for the diagnosis of COVID-19 in the future.

Development and validation of a dried plasma spot LC-MS/MS method for therapeutic monitoring of vancomycin and comparison with enzyme-multiplied immunoassay.

Vancomycin is used as an antimicrobial agent for the treatment of severe gram-positive infections. The importance of therapeutic monitoring of antimicrobials has led to the development of more specific sample preparation techniques capable of identifying with accuracy the concentration of this substance in the organism. An aliquot of 10 µl of plasma was transferred to Whatman 903 paper and dried at room temperature. The extraction method was performed by cutting and transferring the paper to a microtube and adding sodium phosphate buffer and internal standard. The mixture was shaken and centrifuged, and a 5-µl aliquot was injected into the analytical system. The optimization of the main parameters that can influence the extraction efficiency was performed using multivariate approaches to obtain the best conditions. The method developed was validated, providing coefficients of determination higher than 0.994 and a lower limit of quantification of 1 mg/L. Within- and between-run precision ranged from 11.4 to 17.30% and from 6.65 to 13.51%, respectively. This method was successfully applied to 75 samples of patients undergoing vancomycin therapy. The method was rapid, simple, and environmentally friendly with satisfactory analytical performance and was advantageous over the laborious and time-consuming methodologies used in therapeutic drug monitoring routine analyses.

Chitosan/genipin modified electrode for voltammetric determination of interleukin-6 as a biomarker of sepsis.

Ultrasensitive electroanalytical monitoring of interleukin-6 levels in serum samples has emerged as a valuable tool for the early diagnosis of inflammatory diseases. Despite its advantages, there is a lack of strategies for the label-free voltammetric determination of cytokines. Here, a novel chitosan/genipin modified fluorine tin oxide electrode was developed providing an in-situ hydrogel formation (FTO/CSG). This platform was applied for the detection of interleukin-6, a major pro-inflammatory cytokine. Transmission electron microscopy (TEM), cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS) indicated genipin serves as an efficient green cross-linker to build the immunosensing platform (FTO/CSG/anti-IL-6). EIS showed an increase in charge transfer resistance from 326 to 1360 kΩ after the immobilization of anti-IL-6 antibodies. By square wave voltammetry, this method achieved a detection limit of 0.03 pg mL-1 with a wide linear range of 0.05-1000 pg mL-1. Additionally, it displayed a high selectivity index when tested in the presence of three inflammatory cytokines as interfering proteins: IL-12, IL-1ß, and TNF-α. The sample matrix effect showed a peak current variation lower than 5 %. The novel method was applied for the quantification of IL-6 in serum samples of septic mice. No statistical differences were observed between the standard ELISA and the proposed method using a confidence level of 95 %.

Biochar: An environmentally friendly platform for construction of a SARS-CoV-2 electrochemical immunosensor.

Waste management is a key feature to ensure sustainable consumption and production patterns, and to combat the impacts of climate change. In this scenario, the production of biochar from different biomasses results in environmental and economic advantages. In this study, biochar was produced from sugarcane bagasse pyrolysis, to immobilize biomolecules, in order to assemble an electrochemical immunosensor to detect antibodies against SARS-CoV-2. For this, screen-printed carbon electrodes (SPCE) were modified with a dispersion of biochar and used to immobilize the receptor-binding-domain (RBD) against virus S-protein, through EDC/NHS crosslinking reaction. Under the best set of experimental conditions, negative and positive serum samples responses distinguished based on a cutoff value of 82.3 %, at a 95 % confidence level. The immunosensor showed selective behavior to antibodies against yellow fever and its performance was stable up to 7 days of storage. Therefore, biochar yielded from sugarcane bagasse is an ecofriendly material that can be used as a platform to immobilize biomolecules for construction of electrochemical biosensors.