Selection, characterization, and application of DNA aptamers for detection of Mycobacterium tuberculosis secreted protein MPT64

Abstract Rapid detection of Mycobacterium tuberculosis (Mtb), an etiological agent of tuberculosis (TB), is important for global control of this disease. Aptamers have emerged as a potential rival for antibodies in therapeutics, diagnostics and biosensing due to their inherent characteristics. The aim of the current study was to select and characterize single-stranded DNA aptamers against MPT64 protein, one of the predominant secreted proteins of Mtb pathogen. Aptamers specific to MPT64 protein were selected in vitro using systematic evolution of ligands through exponential enrichment (SELEX) method. The selection was started with a pool of ssDNA library with randomized 40-nucleotide region. A total of 10 cycles were performed and seventeen aptamers with unique sequences were identified by sequencing. Dot Blot analysis was performed to monitor the SELEX process and to conduct the preliminary tests on the affinity and specificity of aptamers. Enzyme linked oligonucleotide assay (ELONA) showed that most of the aptamers were specific to the MPT64 protein with a linear correlation of R2 = 0.94 for the most selective. Using Surface Plasmon Resonance (SPR), dissociation equilibrium constant KD of 8.92 nM was obtained. Bioinformatics analysis of the most specific aptamers revealed the existence of a conserved as well as distinct sequences and possible binding site on MPT64. The specificity was determined by testing non-target ESAT-6 and CFP-10. Negligible cross-reactivity confirmed the high specificity of the selected aptamer. The selected aptamer was further tested on clinical sputum samples using ELONA and had sensitivity and specificity of 91.3% and 90%, respectively. Microscopy, culture positivity and nucleotide amplification methods were used as reference standards. The aptamers studied could be further used for the development of medical diagnostic tools and detection assays for Mtb

CFD Modeling of Chamber Filling in a Micro- Biosensor for Protein Detection

Tuberculosis (TB) remains one of the main causes of human death around the globe. The mortality rate for patients infected with active TB goes beyond 50% when not diagnosed. Rapid and accurate diagnostics coupled with further prompt treatment of the disease is the cornerstone for controlling TB outbreaks. To reduce this burden, the existing gap between detection and treatment must be addressed, and dedicated diagnostic tools such as biosensors should be developed. A biosensor is a sensing micro-device that consists of a biological sensing element and a transducer part to produce signals in proportion to quantitative information about the binding event. The micro-biosensor cell considered in this investigation is designed to operate based on aptamers as recognition elements against Mycobacterium tuberculosis secreted protein MPT64, combined in a microfluidic-chamber with inlet and outlet connections. The microfluidic cell is a miniaturized platform with valuable advantages such as low cost of analysis with low reagent consumption, reduced sample volume, and shortened processing time with enhanced analytical capability. The main purpose of this study is to assess the flooding characteristics of the encapsulated microfluidic cell of an existing micro-biosensor using Computational Fluid Dynamics (CFD) techniques. The main challenge in the design of the microfluidic cell lies in the extraction of entrained air bubbles, which may remain after the filling process is completed, dramatically affecting the performance of the sensing element. In this work, a CFD model was developed on the platform ANSYS-CFX using the finite volume method to discretize the domain and solving the Navier–Stokes equations for both air and water in a Eulerian framework. Second-order space discretization scheme and second-order Euler Backward time discretization were used in the numerical treatment of the equations. For a given inlet–outlet diameter and dimensions of an in-house built cell chamber, different inlet liquid flow rates were explored to determine an appropriate flow condition to guarantee an effective venting of the air while filling the chamber. The numerical model depicted free surface waves as promoters of air entrainment that ultimately may explain the significant amount of air content in the chamber observed in preliminary tests after the filling process is completed. Results demonstrated that for the present design, against the intuition, the chamber must be filled with liquid at a modest flow rate to minimize free surface waviness during the flooding stage of the chamber

Role of viruses in the development of breast cancer

The most common cancer worldwide among women is breast cancer. The initiation, promotion, and progression of this cancer result from both internal and external factors. The International Agency for Research on Cancer stated that 18-20% of cancers are linked to infection, and the list of definite, probable, and possible carcinogenic agents is growing each year. Among them, biological carcinogens play a significant role. In this review, data covering infection-associated breast and lung cancers are discussed and presented as possible involvements as pathogens in cancer. Because carcinogenesis is a multistep process with several contributing factors, we evaluated to what extent infection is significant, and concluded that members of the herpesvirus, polyomavirus, papillomavirus, and retrovirus families definitely associate with breast cancer. Detailed studies of viral mechanisms support this conclusion, but have presented problems with experimental settings. It is apparent that more effort needs to be devoted to assessing the role of these viruses in carcinogenesis, by characterizing additional confounding and synergistic effects of carcinogenic factors. We propose that preventing and treating infections may possibly stop or even eliminate certain types of cancers

Role of viruses in the development of breast cancer

The most common cancer worldwide among women is breast cancer. The initiation, promotion, and progression of this cancer result from both internal and external factors. The International Agency for Research on Cancer stated that 18-20% of cancers are linked to infection, and the list of definite, probable, and possible carcinogenic agents is growing each year. Among them, biological carcinogens play a significant role. In this review, data covering infection-associated breast and lung cancers are discussed and presented as possible involvements as pathogens in cancer. Because carcinogenesis is a multistep process with several contributing factors, we evaluated to what extent infection is significant, and concluded that members of the herpesvirus, polyomavirus, papillomavirus, and retrovirus families definitely associate with breast cancer. Detailed studies of viral mechanisms support this conclusion, but have presented problems with experimental settings. It is apparent that more effort needs to be devoted to assessing the role of these viruses in carcinogenesis, by characterizing additional confounding and synergistic effects of carcinogenic factors. We propose that preventing and treating infections may possibly stop or even eliminate certain types of cancers

Refractive Index Sensor by Interrogation of Etched MgO Nanoparticle-Doped Optical Fiber Signature

International audienc

An aptasensor for the detection of Mycobacterium tuberculosis secreted immunogenic protein MPT64 in clinical samples towards tuberculosis detection

This work presents experimental results on detection of Mycobacterium tuberculosis secreted protein MPT64 using an interdigitated electrode (IDE) which acts as a platform for capturing an immunogenic protein and an electrochemical impedance spectroscopy (EIS) as a detection technique. The assay involves a special receptor, single stranded DNA (ssDNA) aptamer, which specifically recognizes MPT64 protein. The ssDNA immobilization on IDE was based on a co-adsorbent immobilization at an optimized ratio of a 1/100 HS-(CH6)(6)-OP(O)(2)O-(CH2CH2O)(6)-5'-TTTTT-aptamer-3'/6-mercaptohexanol. The optimal sample incubation time required for a signal generation on an aptamer modified IDE was found to be at a range of 15-20 min. Atomic Force Microscopy (AFM) results confirmed a possible formation of an aptamer - MPT64 complex with a 20 nm roughness on the IDE surface vs. 4.5 nm roughness for the IDE modified with the aptamer only. A limit of detection for the EIS aptasensor based on an IDE for the detection of MPT64 in measurement buffer was 4.1 fM. The developed EIS aptasensor was evaluated on both serum and sputum clinical samples from the same TB (-) and TB (+) patients having a specificity and sensitivity for the sputum sample analysis 100% and 76.47%, respectively, and for the serum sample analysis 100% and 88.24%, respectively. The developed aptasensor presents a sensitive method for the TB diagnosis with the fast detection time

Fiber Optic Refractive Index Distributed Multi-Sensors by Scattering-Level Multiplexing With MgO Nanoparticle-Doped Fibers

© 2020 IEEE. Personal use of this material is permitted. Permissíon from IEEE must be obtained for all other uses, in any current or future media, including reprinting/republishing this material for advertisíng or promotional purposes, creating new collective works, for resale or redistribution to servers or lists, or reuse of any copyrighted component of this work in other works.[EN] In this work, we present the architecture of a multiplexed refractive index (RI) sensing system based on the interrogation of Rayleigh backscattering. The RI sensors are fabricated by fiber wet-etching of a high-scattering MgO nanoparticle-doped fiber, without the need for a reflector or plasmonic element. Interrogation is performed by means of optical backscatter reflectometry(OBR), which allows a detection with a millimeter-level spatial resolution. Multiplexing consists of a simultaneous scan of multiple fibers, achieved by means of scattering-level multiplexing (SLMux) concept, which uses the backscattered power level in each location as a diversity element. The sensors fabricated have sensitivity in the order of 0.473-0.568 nm/RIU (in one sensing point) and have been simultaneously detected together with a distributed temperature sensing element for multi-parameter measurement. An experimental setup has been prepared to demonstrate the capability of each sensing region to operate without cross-talk, while operating multi-fiber detection.This work was supported in part by the ORAU Programme at Nazarbayev University (LIFESTART and FOSTHER Grants), in part by the Agence Nationale de la Recherche (ANR) Project NanoSlim under Grant ANR-17-17-CE08-0002, in part by the National Natural Science Foundation for Excellent Youth Foundation of China under Grant 61722505, in part by the Key Program of Guangdong Natural Science Foundation under Grant 2018B030311006, and in part by The Spanish Ministry of Economy and Competitiveness under Grant DIMENSION TEC2017 88029-R. The associate editor coordinating the review of this article and approving it for publication was Prof. Marco Petrovich.Ayupova, T.; Shaimerdenova, M.; Korganbayev, S.; Sypabekova, M.; Bekmurzayeva, A.; Blanc, W.; Sales Maicas, S.... (2020). Fiber Optic Refractive Index Distributed Multi-Sensors by Scattering-Level Multiplexing With MgO Nanoparticle-Doped Fibers. IEEE Sensors Journal. 20(5):2504-2510. https://doi.org/10.1109/JSEN.2019.2953231S2504251020

Fiber Bragg Gratings for Medical Applications and Future Challenges: A Review

[EN] In the last decades, fiber Bragg gratings (FBGs) have become increasingly attractive to medical applications due to their unique properties such as small size, biocompatibility, immunity to electromagnetic interferences, high sensitivity and multiplexing capability. FBGs have been employed in the development of surgical tools, assistive devices, wearables, and biosensors, showing great potentialities for medical uses. This paper reviews the FBG-based measuring systems, their principle of work, and their applications in medicine and healthcare. Particular attention is given to sensing solutions for biomechanics, minimally invasive surgery, physiological monitoring, and medical biosensing. Strengths, weaknesses, open challenges, and future trends are also discussed to highlight how FBGs can meet the demands of next-generation medical devices and healthcare system.This work was supported in part by INAIL (the Italian National Institute for Insurance against Accident at Work), through the BRIC (Bando ricerche in collaborazione) 2018 SENSE-RISC (Sviluppo di abiti intelligENti Sensorizzati per prevenzione e mitigazione di Rischi per la SiCurezza dei lavoratori) Project under Grant ID10/2018, in part by the UCBM (Universita Campus Bio-Medico di Roma) under the University Strategic HOPE (HOspital to the PatiEnt) Project, in part by the EU Framework Program H2020-FETPROACT-2018-01 NeuHeart Project under Grant GA 824071, by FCT/MEC (Fundacao para a Ciencia e Tecnologia) under the Projects UIDB/50008/2020 - UIDP/50008/2020, and by REACT (Development of optical fiber solutions for Rehabilitation and e-Health applications) FCT-IT-LA scientific action.Lo Presti, D.; Massaroni, C.; Leitao, CSJ.; Domingues, MDF.; Sypabekova, M.; Barrera, D.; Floris, I.... (2020). Fiber Bragg Gratings for Medical Applications and Future Challenges: A Review. IEEE Access. 8:156863-156888. https://doi.org/10.1109/ACCESS.2020.3019138S156863156888

Electrochemical Aptasensor using Optimized Surface Chemistry for the Detection of Mycobacterium tuberculosis Secreted Protein MPT64 in Human Serum

Tuberculosis (TB) remains one of the leading causes of mortality worldwide. There is a great need for the development of diagnostic tests, which are reliable, sensitive, stable, and low cost to enable early diagnosis of TB in communities with scarce resources. This study reports the optimi-zation and evaluation of a synthetic receptor, an aptamer, for the detection of the secreted pro-tein MPT64, which is a highly immunogenic polypeptide of Mycobacterium tuberculosis, a causa-tive agent of TB. The study investigates combinatorial effects of an aptamer linker and a co-adsorbent onto a gold electrode for optimal binding efficiency and reduced non-specific interac-tions for label-free detection of MPT64 using electrochemical impedance spectroscopy. Two types of co-adsorbents and two types of aptamer linkers were studied and high specificity and sensitivity to MPT64 was observed for a surface prepared with a thiol PEGylated aptamer HS-(CH2)6-OP(O)2O-(CH2CH2O)6-TTTTT-aptamer and 6-mercaptohexanol in a ratio of 1:100. The developed aptamer-based sensor was successfully used with spiked human serum sample with a limit of detection of 81 pM. This work demonstrates the use of the MPT64 aptamer as a lower cost, more sustainable and stable alternative of antibodies for the development of point-of-care TB biosensors decreasing the detection time from several days or hours to thirty minutes