RESUMO
Drug-induced liver injury (DILI) is a major challenge in clinical medicine and drug development. There is a need for rapid diagnostic tests, ideally at point-of-care. MicroRNA 122 (miR-122) is an early biomarker for DILI which is reported to increase in the blood before standard-of-care markers such as alanine aminotransferase activity. We developed an electrochemical biosensor for diagnosis of DILI by detecting miR-122 from clinical samples. We used electrochemical impedance spectroscopy (EIS) for direct, amplification free detection of miR-122 with screen-printed electrodes functionalised with sequence specific peptide nucleic acid (PNA) probes. We studied the probe functionalisation using atomic force microscopy and performed elemental and electrochemical characterisations. To enhance the assay performance and minimise sample volume requirements, we designed and characterised a closed-loop microfluidic system. We presented the EIS assay's specificity for wild-type miR-122 over non-complementary and single nucleotide mismatch targets. We successfully demonstrated a detection limit of 50 pM for miR-122. Assay performance could be extended to real samples; it displayed high selectivity for liver (miR-122 high) comparing to kidney (miR-122 low) derived samples extracted from murine tissue. Finally, we successfully performed an evaluation with 26 clinical samples. Using EIS, DILI patients were distinguished from healthy controls with a ROC-AUC of 0.77, a comparable performance to qPCR detection of miR-122 (ROC-AUC: 0.83). In conclusion, direct, amplification free detection of miR-122 using EIS was achievable at clinically relevant concentrations and in clinical samples. Future work will focus on realising a full sample-to-answer system which can be deployed for point-of-care testing.
Assuntos
Técnicas Biossensoriais , Doença Hepática Induzida por Substâncias e Drogas , MicroRNA Circulante , MicroRNAs , Humanos , Camundongos , Animais , Técnicas Biossensoriais/métodos , Testes Imediatos , Sistemas Automatizados de Assistência Junto ao Leito , Doença Hepática Induzida por Substâncias e Drogas/diagnóstico , Técnicas Eletroquímicas , MicroRNAs/análiseRESUMO
This study presents a new strategy and level of mechanistic understanding for ultrasensitive detection of short, non-coding RNAs without target amplification or chemical modification using electrochemical biosensors. Electrochemical impedance spectroscopy (EIS) has been used for probe target interaction detection because of its high utility for sensitive and label-free measurements of the nucleic acid targets as a result of hybridisation. EIS measurements of different probe target combinations in a range of spatial orientations and sequence overlaps showed that bringing the target overhangs closer to the nanometer proximity of the electrode surface improved the EIS signal significantly. Systematic investigations using different lengths of overhangs towards the electrode surface revealed proportionally higher EIS signals with increasing lengths of the overhangs. Our observations could be explained using the Poisson-Boltzmann and Gouy-Chapman model and followed our experimental modelling. In conclusion, the optimized arrangements of our EIS biosensor system enabled us to detect microRNA-122, a known biomarker for liver injury, as well as three common isoforms to a 1 nM (equivalent to 80 fmole) detection limit. This will enable us to develop solutions for the detection of this important blood biomarker at point of care.
Assuntos
Técnicas Biossensoriais , MicroRNAs , Técnicas Biossensoriais/métodos , Espectroscopia Dielétrica , Técnicas Eletroquímicas , Eletrodos , Limite de Detecção , Hibridização de Ácido NucleicoRESUMO
We present the development of electrochemical impedance spectroscopy (EIS)-based biosensors for sensitive detection of SARS-CoV-2 RNA using multi-valent binding. By increasing the number of probe-target binding events per target molecule, multi-valent binding is a viable strategy for improving the biosensor performance. As EIS can provide sensitive and label-free measurements of nucleic acid targets during probe-target hybridization, we used multi-valent binding to build EIS biosensors for targeting SARS-CoV-2 RNA. For developing the biosensor, we explored two different approaches including probe combinations that individually bind in a single-valent fashion and the probes that bind in a multi-valent manner on their own. While we found excellent biosensor performance using probe combinations, we also discovered unexpected signal suppression. We explained the signal suppression theoretically using inter- and intra-probe hybridizations which confirmed our experimental findings. With our best probe combination, we achieved a LOD of 182 copies/µL (303 aM) of SARS-CoV-2 RNA and used these for successful evaluation of patient samples for COVID-19 diagnostics. We were also able to show the concept of multi-valent binding with shorter probes in the second approach. Here, a 13-nt-long probe has shown the best performance during SARS-CoV-2 RNA binding. Therefore, multi-valent binding approaches using EIS have high utility for direct detection of nucleic acid targets and for point-of-care diagnostics.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , COVID-19/diagnóstico , RNA Viral/genética , Hibridização de Ácido NucleicoRESUMO
Drug-induced liver injury (DILI) results in over 100 000 hospital attendances per year in the UK alone and is a leading cause for the post-marketing withdrawal of new drugs, leading to significant financial losses. MicroRNA-122 (miR-122) has been proposed as a sensitive DILI marker although no commercial applications are available yet. Extracellular blood microRNAs (miRNAs) are promising clinical biomarkers but their measurement at point of care remains time-consuming, technically challenging, and expensive. For circulating miRNA to have an impact on healthcare, a key challenge to overcome is the development of rapid and reliable low-cost sample preparation. There is an acknowledged issue with miRNA stability in the presence of hemolysis and platelet activation, and no solution has been demonstrated for fast and robust extraction at the site of blood draw. Here, we report a novel microfluidic platform for the extraction of circulating miR-122 from blood enabled by a vertical approach and gravity-based bubble mixing. The performance of this disposable cartridge was verified by standard quantitative polymerase chain reaction analysis on extracted miR-122. The cartridge performed equivalently or better than standard bench extraction kits. The extraction cartridge was combined with electrochemical impedance spectroscopy to detect miR-122 as an initial proof-of-concept toward an application in point-of-care detection. This platform enables the standardization of sample preparation and the detection of miRNAs at the point of blood draw and in resource limited settings and could aid the introduction of miRNA-based assays into routine clinical practice.
RESUMO
This paper demonstrates a new and simplified configuration for capillary electrophoresis-amperometric detection (CE-AD) using a paper microfluidic chip incorporating inexpensive wax printing and screen printing based methods and then used for electrophoretic separation and simultaneous in-channel amperometric detection of three clinically relevant neurochemicals in a single run without using any decouplers. Detection of neurochemicals e.g., dopamine, epinephrine and serotonin is crucial for early prediction of neurological disorders including Parkinson's, Alzheimer's, dementia, as well as progressive neuro-psychiatric conditions such as depression, anxiety, as well as certain cardiovascular diseases. The plasma concentrations of such neurochemicals are as important as those present in cerebrospinal fluid (CSF) and can be useful for rapid and convenient biosensing. However, simultaneous detection of such neurochemicals in a complex mixture such as human serum requires their separation prior to detection. With the developed microchip, separation and detection of the neurochemicals were exhibited within 650 seconds without pre-treatment and the procedure was validated with spiked fetal bovine serum samples. Beside this, the developed paper microfluidic chip has potential to be integrated in point-of-care diagnosis with onsite detection ability. Moreover, the use of a straight channel capillary, a screen-printed carbon electrode without decoupler, in-channel amperometric detection and low sample volume requirements (2 µL) are shown as additional advantages.
RESUMO
An amperometric biosensor has been developed for highly efficient and sensitive detection of catechol using Prussian blue (PB)-coated nickel oxide (NiO) nanoparticles (NPs) as a matrix for the immobilization of tyrosinase enzyme. The NiO NPs were synthesized by sol-gel method using sodium dodecyl sulphate as anionic surfactant and the surface of the synthesized NiO NPs was modified with PB to enhance electrocatalytic activity and to prevent surface aggregation. After confirmation of successful synthesis of the PB-NiO NPs from transmission electron microscopy (TEM) and energy dispersive X-ray spectroscopic (EDS) studies, the prepared NPs were deposited onto a working electrode of a commercially available screen printed carbon electrode (SPCE) substrate. The tyrosinase enzyme was covalently immobilized onto the PB-NiO deposited SPCE for selective detection and estimation of catechol through electrochemical methods via cyclic voltammetry (CV) and chronoamperometric techniques. The functionalization of tyrosinase on the electrode surface was verified by atomic force microscopy (AFM) and scanning electron microscopic (SEM) techniques and the electrochemical response studies of the proposed biosensor showed high sensitivity of 0.954 µA/µM for catechol in a wide linear range (1 - 50 µM) with low detection limit (LOD) of 0.087 µM. The developed sensor also exhibited a fast response time of 27 s and decent selectivity for catechol detection.
Assuntos
Técnicas Biossensoriais/métodos , Catecóis/análise , Ferrocianetos/química , Nanopartículas/química , Níquel/química , Técnicas Biossensoriais/instrumentação , Carbono/química , Catecóis/química , Eletroquímica , Eletrodos , Impressão , Propriedades de SuperfícieRESUMO
A dopamine biosensor has been developed using nickel oxide nanoparticles (NPs) and tyrosinase enzyme conjugate. Nickel oxide (NiO) NPs were synthesized by sol-gel method using anionic surfactant, sodium dodecyl sulphate (SDS), as template to control the size of synthesized nanoparticles. The structural and morphological studies of the prepared NPs were carried out using X-ray diffraction (XRD), transmission electron microscopy (TEM) and dynamic light scattering (DLS) techniques. Afterwards, tyrosinase enzyme molecules were adsorbed on NiO NPs surface and enzyme coated NPs were deposited on indium tin oxide (ITO) coated flexible polyethylene terephthalate (PET) substrate by solution casting method. The formation of enzyme-NPs conjugate was investigated by atomic force microscopy (AFM) and Fourier transform infrared spectroscopy (FTIR) techniques and used in selective detection and estimation of neurochemical dopamine by electrochemical method. The fabricated Tyrosinase/NiO/ITO electrode exhibits high sensitivity of 60.2nA/µM in linear detection range (2-100µM) with a detection limit of 1.038µM. The proposed sensor had a response time of 45s, long shelf life (45 days) with good reproducibility and selectivity in presence of interfering substances and was validated with real samples. The tyrosinase enzyme functionalized NiO platform has good bio-sensing efficacy and can be used in detection of other catecholamines and phenolic neurochemicals.
Assuntos
Técnicas Biossensoriais/métodos , Dopamina/sangue , Técnicas Eletroquímicas/métodos , Nanopartículas/química , Níquel/química , Agaricus/enzimologia , Animais , Bovinos , Enzimas Imobilizadas/química , Limite de Detecção , Monofenol Mono-Oxigenase/química , Nanopartículas/ultraestrutura , Polietilenotereftalatos/química , Compostos de Estanho/químicaRESUMO
In this paper, an egg-shell membrane has been used for efficient immobilization and stabilization of glucose oxidase. This membrane was used for developing a simple and reusable method for estimation of glucose in biological samples. The proposed sensor was effectively used in a wide glucose concentration range (1 - 1000 mM) with fast response time of 70 s for higher concentrations and 120 s for lower concentrations. The results of response study for the fabricated sensor show limit of detection of 4.761 mM with high sensitivity over the entire concentration range (1 - 1000 mM). Most interestingly, the membrane used in the fabricated sensor could be repeatedly used for glucose analysis 150 times and it exhibited a shelf-life of more than 6 weeks. The proposed sensor was also demonstrated for estimation of glucose in human blood samples.
Assuntos
Técnicas Biossensoriais/métodos , Membrana Corioalantoide/química , Casca de Ovo , Enzimas Imobilizadas/química , Glucose Oxidase/química , Glucose/análise , Animais , Técnicas Biossensoriais/instrumentação , Calibragem , Desenho de Equipamento , Software , Fatores de TempoRESUMO
In this paper, we present results of the studies related to fabrication of a rare earth metal oxide based efficient biosensor using an interface based on hydrothermally prepared nanostructured thulium oxide (n-Tm2O3). A colloidal solution of prepared nanorods has been electrophoretically deposited (EPD) onto an indium-tin-oxide (ITO) glass substrate. The n-Tm2O3 nanorods are found to provide improved sensing characteristics to the electrode interface in terms of electroactive surface area, diffusion coefficient, charge transfer rate constant and electron transfer kinetics. The structural and morphological studies of n-Tm2O3 nanorods have been carried out by X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared (FTIR) spectroscopic techniques. This interfacial platform has been used for fabrication of a total cholesterol biosensor by immobilizing cholesterol esterase (ChEt) and cholesterol oxidase (ChOx) onto a Tm2O3 nanostructured surface. The results of response studies of the fabricated ChEt-ChOx/n-Tm2O3/ITO bioelectrode show a broad linear range of 8-400 mg dL(-1), detection limit of 19.78 mg (dL cm(-2))(-1), and high sensitivity of 0.9245 µA (mg per dL cm(-2))(-1) with a response time of 40 s. Further, this bioelectrode has been utilized for estimation of total cholesterol with negligible interference (3%) from analytes present in human serum samples. The utilization of this n-Tm2O3 modified electrode for enzyme-based biosensor analysis offers an efficient strategy and a novel interface for application of the rare earth metal oxide materials in the field of electrochemical sensors and bioelectronic devices.
Assuntos
Colesterol Oxidase/metabolismo , Nanoestruturas/química , Esterol Esterase/metabolismo , Túlio/química , Técnicas Biossensoriais , Colesterol/sangue , Colesterol Oxidase/química , Técnicas Eletroquímicas , Eletrodos , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Vidro/química , Humanos , Cinética , Nanotubos/química , Esterol Esterase/química , Compostos de Estanho/químicaRESUMO
The ultrafine monodispersed cuprous oxide (Ufm-Cu(2)O) nanoparticles have been successfully synthesized by a facile wet chemical method using poly-N-vinylpyrrolidone (PVP) as a capping agent. This colloidal solution of Ufm-Cu(2)O and chitosan (CS) is electrophoretically deposited (EPD) onto the indium tin-oxide (ITO) glass substrate. Thus synthesized nanocomposite has been characterized by X-ray powder diffraction (XRD, â¼6 nm), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and Fourier transform infrared (FTIR) spectroscopic techniques. This novel biomedical nanocomposite platform has been explored to fabricate a cholesterol biosensor by immobilizing cholesterol esterase (ChEt) and cholesterol oxidase (ChOx) onto Ufm-Cu(2)O-CS/ITO electrode surface. The seed germination tests of these biomaterials (Ufm-Cu(2)O-CS nanocomposite and ChOx-ChEtUfm-CuO(2)-CS nanobiocomposite), conducted using the disc diffusion method, reveal strong activity against the common pathogens and crops, indicating biocompatibility of the nanocomposite. Under optimized conditions, the linearity between the current response and the cholesterol concentration has been obtained in the range of 10-450 mg/dL, with detection limit of 15.9 mg/dL cm(-2) and a high sensitivity of 0.895 µA/(mg/dL cm(-2)). The proposed biocompatible ChEt-ChOx/Ufm-Cu(2)O-CS/ITO bioelectrode shows fast response time (<5 s), good reproducibility, and long-term stability. This biocompatible biosensor has been used to determine the total cholesterol levels in human serum samples. Investigated antimicrobial activities of bienzyme-functionalized Ufm-Cu(2)O-CS nanocomposite are the potential platform for biomedical applications.
Assuntos
Materiais Biocompatíveis , Quitosana/química , Cobre/química , Nanocompostos , Concentração de Íons de Hidrogênio , Espectroscopia de Infravermelho com Transformada de Fourier , Difração de Raios XRESUMO
The nanostructured rare earth metal oxide (samarium oxide, n-Sm2O3) nanorods, prepared using a forced hydrolysis technique, have been electrophoretically deposited (EPD) onto an indium-tin-oxide (ITO) glass substrate. This novel platform has been utilized for co-immobilization of monoclonal antibodies of aflatoxin B1 (Ab-AFB1) and bovine serum albumin (BSA) via electrostatic interactions for food toxin (AFB1) detection. Thus prepared n-Sm2O3 nanorods have been characterized by X-ray diffraction (XRD), atomic force microscopy (AFM), transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared (FTIR) spectroscopic techniques. The results of electrochemical response studies of the BSA/Ab-AFB1/n-Sm2O3/ITO immunoelectrode obtained as a function of aflatoxin concentration reveal a linearity of 10-700 pg mL-1, a detection limit of 57.82 pg mL-1 cm-2, a response time of 5 s and a sensitivity of 48.39 µA pg-1 mL-1 cm-2 with a regression coefficient of 0.961. The association constant (Ka) for antigen-antibody interactions obtained is 47.9 pg mL-1, which indicates high affinity of antibodies towards the antigen (AFB1). The application of n-Sm2O3 modified electrode for immunosensor analysis offers a novel platform and efficient strategy for the application of rare earth metal oxide materials in bioelectronics.