RESUMEN
Reagentless molecular-imprinted polymer (MIP) electrochemical biosensors can offer the next generation of biosensing platforms for the detection of biomarkers owing to their simplicity, cost-efficacy, tunability, robustness, and accuracy. In this work, a novel combination of Prussian blue (PB), coated as an embedded redox probe on a gold working electrode (GWE), and a signal-off MIP assay has been proposed in an electrochemical format for the detection of troponin I (TnI) in biofluids. TnI is a variant exclusive to heart muscles, and its elevated level in the bloodstream is indicative of acute myocardial infarction (AMI). The proposed lab-manufactured PB/MIP electrochemical biosensor, consisting of a simple signal-off MIP assay and a PB redox probe embedded on the GWE surface, is the first of its kind that allows for reagentless, label-free, and single-step electrochemical biosensing of proteins. The preparation steps of the biosensor were fully characterized by cyclic voltammetry (CV), atomic force microscopy (AFM), and Raman spectroscopy. Finally, the performance of the optimized biosensor was investigated through the determination of various concentrations of TnI, ranging from 10 to 100 pg mL-1 within 5 min, in serum and plasma with limits of detection less than 3.6 pg mL-1, and evaluation of selectivity towards TnI using some relevant proteins that exist in biofluids with higher concentrations.
Asunto(s)
Técnicas Biosensibles , Técnicas Electroquímicas , Oro , Polímeros Impresos Molecularmente , Troponina I , Humanos , Técnicas Biosensibles/métodos , Técnicas Electroquímicas/métodos , Técnicas Electroquímicas/instrumentación , Electrodos , Ferrocianuros/química , Oro/química , Límite de Detección , Polímeros Impresos Molecularmente/química , Polímeros/química , Troponina I/sangre , Troponina I/análisisRESUMEN
Novel biomaterials for bio- and chemical sensing applications have gained considerable traction in the diagnostic community with rising trends of using biocompatible and lowly cytotoxic material. Hydrogel-based electrochemical sensors have become a promising candidate for their swellable, nano-/microporous, and aqueous 3D structures capable of immobilizing catalytic enzymes, electroactive species, whole cells, and complex tissue models, while maintaining tunable mechanical properties in wearable and implantable applications. With advances in highly controllable fabrication and processability of these novel biomaterials, the possibility of bio-nanocomposite hydrogel-based electrochemical sensing presents a paradigm shift in the development of biocompatible, "smart," and sensitive health monitoring point-of-care devices. Here, recent advances in electrochemical hydrogels for the detection of biomarkers in vitro, in situ, and in vivo are briefly reviewed to demonstrate their applicability in ideal conditions, in complex cellular environments, and in live animal models, respectively, to provide a comprehensive assessment of whether these biomaterials are ready for point-of-care translation and biointegration. Sensors based on conductive and nonconductive polymers are presented, with highlights of nano-/microstructured electrodes that provide enhanced sensitivity and selectivity in biocompatible matrices. An outlook on current challenges that shall be addressed for the realization of truly continuous real-time sensing platforms is also presented.
Asunto(s)
Técnicas Biosensibles , Dispositivos Electrónicos Vestibles , Animales , Hidrogeles/química , Polímeros , Materiales Biocompatibles/química , NanogelesRESUMEN
Colorimetric readout for the detection of infectious diseases is gaining traction at the point of care/need owing to its ease of analysis and interpretation, and integration potential with highly specific loop-mediated amplification (LAMP) assays. However, coupling colorimetric readout with LAMP is rife with challenges including, rapidity, inter-user variability, colorimetric signal quantification, and user involvement in sequential steps of the LAMP assay, hindering its application. To address these challenges, for the first time, we propose a remotely smartphone-operated automated setup consisting of (i) an additively manufactured microfluidic cartridge, (ii) a portable reflected-light imaging setup with controlled epi-illumination (PRICE) module, and (iii) a control and data analysis module. The microfluidic cartridge facilitates sample collection, lysis, mixing of amplification reagents stored on-chip, and subsequent isothermal heating for initiation of amplification in a novel way by employing tunable elastomeric chambers and auxiliary components (heaters and linear actuators). PRICE offers a new imaging setup that captures the colorimetric change of the amplification media over a plasmonic nanostructured substrate in a controlled and noise-free environment for rapid minute-scale nucleic acid detection. The control and data analysis module employs microprocessors to automate cartridge operation in tandem with the imaging module. The different device components were characterized individually and finally, as a proof of concept, SARS-CoV-2 wild-type RNA was detected with a turnaround time of 13 minutes, showing the device's clinical feasibility. The suggested automated device can be adopted in future iterations for other detection and molecular assays that require sequential fluid handling steps.
Asunto(s)
COVID-19 , Humanos , COVID-19/diagnóstico , Colorimetría , Microfluídica , SARS-CoV-2 , BioensayoRESUMEN
Cryptosporidium parvum is a high-risk and opportunistic waterborne parasitic pathogen with highly infectious oocysts that can survive harsh environmental conditions for long periods. Current state-of-the-art methods are limited to lengthy imaging and antibody-based detection techniques that are slow, labor-intensive, and demand trained personnel. Therefore, the development of new sensing platforms for rapid and accurate identification at the point-of-care (POC) is essential to improve public health. Herein, we propose a novel electrochemical microfluidic aptasensor based on hierarchical 3D gold nano-/microislands (NMIs), functionalized with aptamers specific to C. parvum. We used aptamers as robust synthetic biorecognition elements with a remarkable ability to bind and discriminate among molecules to develop a highly selective biosensor. Also, the 3D gold NMIs feature a large active surface area that provides high sensitivity and a low limit of detection (LOD), especially when they are combined with aptamers,. The performance of the NMI aptasensor was assessed by testing the biosensor's ability to detect different concentrations of C. parvum oocysts spiked in different sample matrices, i.e., buffer, tap water, and stool, within 40 min detection time. The electrochemical measurements showed an acceptable LOD of 5 oocysts mL-1 in buffer medium, as well as 10 oocysts mL-1 in stool and tap water media, over a wide linear range of 10-100,000 oocysts mL-1. Moreover, the NMI aptasensor recognized C. parvum oocysts with high selectivity while exhibiting no significant cross-reactivity to other related coccidian parasites. The specific feasibility of the aptasensor was further demonstrated by the detection of the target C. parvum in patient stool samples. Our assay showed coherent results with microscopy and real-time quantitative polymerase chain reaction, achieving high sensitivity and specificity with a significant signal difference (p < 0.001). Therefore, the proposed microfluidic electrochemical biosensor platform could be a stepping stone for the development of rapid and accurate detection of parasites at the POC.
Asunto(s)
Técnicas Biosensibles , Criptosporidiosis , Cryptosporidium parvum , Cryptosporidium , Animales , Humanos , Microfluídica , Criptosporidiosis/diagnóstico , Agua , Oligonucleótidos , Oocistos , Oro/químicaRESUMEN
Portable sample-to-answer devices with applications in point-of-care settings have emerged to obviate the necessity of centralized laboratories for biomarker analysis. In this work, a smartphone-operated and additively manufactured multiplexed electrochemical device (AMMED) is presented for the portable detection of biomarkers in blood and saliva. AMMED is comprised of a customized portable potentiostat with a multiplexing feature, a 3D-printed sample collection cartridge to handle three samples of saliva and blood at the same time, a smartphone application to remotely control the potentiostat, and a 3D-printed-based multiplexed microfluidic electrochemical biosensor (test chip). Here, by employing additive manufacturing techniques, a simple, cleanroom-free, and scalable approach was proposed for the fabrication of the test chip. Moreover, these techniques can bring about easy integration of AMMED components. Additionally, the test chip can be compatible with different affinity-based bioassays which can be implemented in a multiplexed manner for detection. The AMMED components were successfully characterized in terms of electrochemical and fluidic performance. Particularly, to demonstrate the biosensing capabilities of the device, the spike protein of the SARS-CoV-2 omicron variant and a well-established aptameric assay were selected as the representative biomarker and the bioassay, respectively. The proposed device accurately and selectively detected the target of interest in a rapid (5 min) and multiplex manner with a dynamic detection range of 1-10 000 pg ml-1 in different media, and the clinical feasibility was assessed by several saliva patient samples. AMMED offers a versatile sample-to-answer platform that can be used for the detection of various biomarkers present in biofluids.