Metal-enhanced fluorescence biosensor integrated in capillary flow-driven microfluidic cartridge for highly sensitive immunoassays.

Point-of-care testing (POCT) for low-concentration protein biomarkers remains challenging due to limitations in biosensor sensitivity and platform integration. This study addresses this gap by presenting a novel approach that integrates a metal-enhanced fluorescence (MEF) biosensor within a capillary flow-driven microfluidic cartridge (CFMC) for the ultrasensitive detection of the Parkinson's disease biomarker, aminoacyl-tRNA synthetase complex interacting multi-functional protein 2 (AIMP-2). Crucial point to this approach is the orientation-controlled immobilization of capture antibody on a nanodimple-structured MEF substrate within the CFMC. This strategy significantly enhances fluorescence signals without quenching, enabling accurate quantification of low-concentration AIMP-2 using a simple digital fluorescence microscope with a light-emitting diode excitation source and a digital camera. The resulting platform exhibits exceptional sensitivity, achieving a limit of detection in the pg/mL range for AIMP-2 in human serum. Additionally, the CFMC design incorporates a capillary-driven passive sample transport mechanism, eliminating the need for external pumps and further simplifying the detection process. Overall, this work demonstrates the successful integration of MEF biosensing with capillary microfluidics for point-of-care applications.

Point-of-Care PCR Assays for COVID-19 Detection.

Molecular diagnostics has been the front runner in the world's response to the COVID-19 pandemic. Particularly, reverse transcriptase-polymerase chain reaction (RT-PCR) and the quantitative variant (qRT-PCR) have been the gold standard for COVID-19 diagnosis. However, faster antigen tests and other point-of-care (POC) devices have also played a significant role in containing the spread of SARS-CoV-2 by facilitating mass screening and delivering results in less time. Thus, despite the higher sensitivity and specificity of the RT-PCR assays, the impact of POC tests cannot be ignored. As a consequence, there has been an increased interest in the development of miniaturized, high-throughput, and automated PCR systems, many of which can be used at point-of-care. This review summarizes the recent advances in the development of miniaturized PCR systems with an emphasis on COVID-19 detection. The distinct features of digital PCR and electrochemical PCR are detailed along with the challenges. The potential of CRISPR/Cas technology for POC diagnostics is also highlighted. Commercial RT-PCR POC systems approved by various agencies for COVID-19 detection are discussed.

A Chemosensor Based on Gold Nanoparticles and Dithiothreitol (DTT) for Acrylamide Electroanalysis.

Rapid and simple electroanalysis of acrylamide (ACR) was feasible by a gold electrode modified with gold nanoparticles (AuNPs) and dithiothreitol (DTT) with enhanced detection sensitivity and selectivity. The roughness of bare gold (Au) increased from 0.03 µm to 0.04 µm when it was decorated with AuNPs. The self-assembly between DTT and AuNPs resulted in a surface roughness of 0.09 µm. The DTT oxidation occurred at +0.92 V. The Au/AuNPs/DTT surface exhibited a surface roughness of 0.24 µm after its exposure to ACR with repeated analysis. SEM imaging illustrated the formation of a polymer layer on the Au/AuNPs/DTT surface. Surface plasmon resonance analysis confirmed the presence of AuNPs and DTT on the gold electrode and the binding of ACR to the electrode's active surface area. The peak area obtained by differential pulse voltammetry was inversely proportional to the ACR concentrations. The limit of detection (LOD) and the limit of quantitation (LOQ) were estimated to be 3.11 × 10-9 M and 1 × 10-8 M, respectively, with wide linearity ranging from 1 × 10-8 M to 1 × 10-3 M. The estimated levels of ACR in potato chips and coffee samples by the sensor were in agreement with those of high-performance liquid chromatography.

Exploring Providencia rettgeri for application to eco-friendly paper based microbial fuel cell.

We report results of the studies relating to improved stability (40 days) of small sized microbial fuel cell (MFC) fabricated using agarose embedded paper-based proton exchange membrane. A fermentative bacterium Providencia rettgeri was isolated from rotten potato slurry and identified by 16S rRNA sequencing. The electroactivity of the bacteria was monitored via chronoamperometric and cyclic voltammetric studies using a three-electrode system which indicated the presence of bacterial redox mediator. The Matrix-Assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF) and UV-Vis absorption spectroscopy provided the evidence that Providencia rettgeri synthesized folate (vitamin B9) during fermentation that was found to act for the first time as a redox mediator in an MFC. The paper based designed MFC fed with Providencia rettgeri yielded open circuit voltage of 787.9 mV with power and current density of 5.02 W/m3 and 11.26 A/m3, respectively when measured across 10 kΩ. The microbial re-chargeable battery comprising of an assembly of parallelly aligned four units of MFCs when connected in series (total 16 MFCs), generated 1.5 V that was used for powering a red-light emitting device (LED).

Amine-Functionalized MoO3@RGO Nanohybrid-Based Biosensor for Breast Cancer Detection.

We report results of studies relating to development of an ultrasensitive, rapid, and label-free biosensor based on molybdenum trioxide (MoO3) anchored onto the reduced graphene oxide (RGO) for breast cancer detection. The human epidermal growth factor receptor-2 (HER-2) secreted in the serum of breast cancer patients was used as an analyte for the detection. The in situ growth of 1D MoO3 onto reduced graphene oxide (RGO), a 2D carbon substrate, was carried out via one-pot low-temperature hydrothermal synthesis. Subsequently, the surface conjugation of the monoclonal antibodies (anti-HER-2) onto the APTES/MoO3@RGO/ITO electrode was conducted via EDC-NHS covalent chemistry. The structural and morphological properties of the MoO3@RGO nanohybrid were investigated using electron microscopy, X-ray diffraction, scanning electron microscopy, transmission electron microscopy and X-ray photoelectron spectroscopic techniques. The surface area of the MoO3@RGO nanohybrid determined via Brauner-Emmett-Teller analysis was found to be 14 times greater than that of the pristine MoO3. The binding kinetics and the electrochemical activity of the developed platform were determined by cyclic voltammetry, differential pulse voltammetry, and impedance spectroscopic techniques. This nanohybrid based immunosensor exhibited improved sensitivity (13 uA mLng-1cm-2) in a broad concentration range (0.001-500 ng mL-1) with a correlation coefficient of 0.98. The limit of detection of this MoO3@RGO nanohybrid based immunosensor was found to be 0.001 ng mL-1. The results obtained via the developed immunosensor for the quantification of serum HER-2 were validated using ELISA.

Biofunctionalized Nanostructured Yttria Modified Non-Invasive Impedometric Biosensor for Efficient Detection of Oral Cancer.

We report results of the studies relating to the development of an efficient biosensor for non-invasive detection of CYFRA-21-1 cancer biomarker. We used a low dielectric constant material (nanostructured yttrium oxide, nY2O3) for the fabrication of the biosensing platform. The nY2O3 was synthesized via solvothermal process and functionalized using 3-aminopropyl triethoxy silane (APTES). Electrophoretic deposition (EPD) of the functionalized nanomaterial (APTES/nY2O3) onto an indium tin oxide (ITO)-coated glass electrode was conducted at a DC potential of 50V for 60s. The EDC-NHS chemistry was used for covalent immobilization of -COOH bearing monoclonal anti-CYFRA-21-1 onto -NH2 groups of APTES/nY2O3/ITO electrode. To avoid the non-specific interaction on the anti-CYFRA-21-1/APTES/nY2O3/ITO immunoelectrode, bovine serum albumin (BSA) was used. X-ray diffraction (XRD), transmission electron microscopy (TEM), and field emission scanning electron microscopy (FESEM) were utilized for structural and morphological studies, whereas Fourier-transform infrared spectroscopy (FTIR) was used for the bonding analysis. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) techniques were used for electrochemical characterization and response studies of fabricated electrodes. The fabricated immunosensor (BSA/anti-CYFRA-21-1/APTES/nY2O3/ITO) exhibited linearity in the range of 0.01-50 ng·mL-1, sensitivity of 226.0 Ω·mL·ng-1, and lower detection limit of 0.01·ng·mL-1. A reasonable correlation was observed between the results obtained using this biosensor and concentration of CYFRA-21-1 measured through ELISA (enzyme-linked immunosorbent assay) technique in salivary samples of oral cancer patients.

Protein functionalised self assembled monolayer based biosensor for colon cancer detection.

We report results of the studies relating to the fabrication of a surface plasmon resonance (SPR) based label-free immunosensor for real-time monitoring of endothelin-1 (ET-1), a colon cancer biomarker. A gold disk modified with a self-assembled monolayer (SAM) of 11-mercaptoundecanoic acid (11-MUA) was functionalised via covalent immobilization of monoclonal anti-ET-1 antibodies using EDC-NHS (1-(3-(dimethylamine)-propyl)-3-ethylcarbodiimide hydrochloride, N-hydroxy succinimide) chemistry. This immunosensing platform (ethanolamine/anti-ET-1/11-MUA/Au) was characterized via atomic force microscopy (AFM), contact angle (CA) and Fourier transform infrared (FT-IR) spectroscopic techniques. The fabricated SPR electrode was further used to detect ET-1 in the broad concentration range 2-100 pg mL-1, with a detection limit of 0.30 pg mL-1 and remarkable sensitivity of 2.18 mo pg-1mL. The adsorption mechanism was studied using monophasic model and the values of association (ka) and dissociation (kd) constants for anti-ET-1 and ET-1 binding were calculated to be 4.4 ±â€¯0.4 × 105 M-1 s-1 and 2.04 ±â€¯0.0003 × 10-3 s-1, respectively. The results obtained via analysis of serum samples of colorectal cancer patients were found to be in good agreement with those obtained from enzyme-linked immunosorbent assay (ELISA) technique. Further, electrochemical studies were performed to prove the efficacy of the fabricated platform as a point of care device for the detection of ET-1.

Electrochemical paper based cancer biosensor using iron oxide nanoparticles decorated PEDOT:PSS.

We report results of the studies relating to the fabrication of a label-free, flexible, light weight and disposable conducting paper based immunosensing platform comprising of poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) and nanostructured iron oxide (nFe2O3@PEDOT:PSS) nanocomposite for detection of carcinoembryonic antigen (CEA), a cancer biomarker. The effect of various solvents such as sorbitol, ethanol, propanol, n-methyl-2-pyrrolidone (NMP) and dimethyl sulfoxide (DMSO) on the electrical conductivity of Whatman filter paper (WP) modified with nFe2O3@PEDOT:PSS/WP was investigated. The electrical conductivity of the PEDOT:PSS/WP electrode was found to be enhanced by two orders of magnitude (from 6.8× 10-4 to 1.92 × 10-2 Scm-1) after its treatment with DMSO. Further, nFe2O3 doped PEDOT:PSS/WP electrode exhibited the electrical conductivity as 2.4 × 10-2 Scm-1. Besides this, the incorporation of iron oxide nanoparticles (nFe2O3) into PEDOT:PSS/WP resulted in improved electrochemical performance and signal stability. This nFe2O3@PEDOT:PSS/WP based platform was used for immobilization of the anti-carcinoembronic antigen (anti-CEA) protein for quantitative estimation of cancer biomarker (CEA). The results of electrochemical response studies revealed that this conducting paper based immunoelectrode had a sensitivity of 10.2 µAng-1mLcm-2 in the physiological range (4-25 ngmL-1) and shelf life of 34 days. Further, the proposed immunoelectrode was validated with conventional ELISA for the detection of CEA in serum samples of cancer patients.

Microfluidics Based Point-of-Care Diagnostics.

Point-of-care (POC) diagnostic devices have been predicted to provide a boon in health care especially in the diagnosis and detection of diseases. POC devices have been found to have many advantages like a rapid and precise response, portability, low cost, and non-requirement of specialized equipment. The major objective of a POC diagnostic research is to develop a chip-based, self-containing miniaturized device that can be used to examine different analytes in complex samples. Further, the integration of microfluidics (MF) with advanced biosensor technologies is likely to result in improved POC diagnostics. This paper presents the overview of the different materials (glass, silicon, polymer, paper) and techniques for the fabrication of MF based POC devices along with their wide range of biosensor applications. Besides this, the authors have presented in brief the challenges that MF is currently facing along with possible solutions that may result in the availability of the accessible, reliable, and cost-efficient technology. The development of these devices requires the combination of developed MF components into POC devices that are user-friendly, sensitive, stable, accurate, low cost, and minimally invasive. These MF based POC devices have tremendous potential in providing improved healthcare including easy monitoring, early detection of disease, and increased personalization.

Effect of Brownian motion on reduced agglomeration of nanostructured metal oxide towards development of efficient cancer biosensor.

We report results of the studies relating to fabrication of nanostructured metal oxide (NMO) based cancer biosensor. With the help of 2D electroactive reduced graphene oxide (RGO), we successfully inhibited the Brownian motion of NMO that led to reduced agglomeration of NMO. The nanostructured hafnium oxide (nHfO2) was used as a model NMO. The reduced agglomeration of nHfO2 was achieved through controlled hydrothermal synthesis and investigated via nanoparticles tracking analysis (NTA). X-ray diffraction (XRD), scanning electron microscopy (SEM) and transmission electron microscope (TEM) techniques were used for phase identification as well as morphological analysis of the synthesized nanohybrid (nHfO2@RGO) material. The 3-aminopropyl triethoxysilane (APTES) was used for the functionalization of nHfO2@RGO and electrophoretic deposition (EPD) technique was used for its deposition onto ITO coated glass electrode. Further, antibodies of cancer biomarker (anti-CYFRA-21-1) were immobilized via EDC-NHS chemistry and Bovine serum albumin (BSA) was used for blocking of the non-specific binding sites. The electrochemical response studies of fabricated immunoelectrode (BSA/anti-CYFRA-21-1/APTES/nHfO2@RGO/ITO) revealed higher sensitivity (18.24µAmLng-1), wide linear detection range (0 to 30ngmL-1), with remarkable lower detection limit (0.16ngmL-1). The obtained results showed good agreement with the concentration of CYFRA-21-1 obtained through enzyme linked immunosorbent assay (ELISA) in saliva samples of oral cancer patients.

Recent advances in carbon based nanosystems for cancer theranostics.

One of the major challenges in our contemporary society is to facilitate healthy life for all human beings. In this context, cancer has become one of the most deadly diseases around the world, and despite many advances in theranostics techniques the treatment of cancer still remains an important problem. With recent advances made in the field of nano-biotechnology, carbon-based nanostructured materials have drawn special attention because of their unique physicochemical properties, giving rise to great potential for the diagnosis and therapy of cancer. This review deals with four different types of carbon allotrope including carbon nanotubes, graphene, fullerenes and nanodiamonds and summarizes the results of recent studies that are likely to have implications in cancer theranostics. We discuss the applications of these carbon allotropes for cancer imaging and drug delivery, hyperthermia, photodynamic therapy and acoustic wave assisted theranostics. We focus on the results of different studies conducted on functionalized/conjugated carbon nanotubes, graphene, fullerenes and nanodiamond based nanostructured materials reported in the literature in the current decade. The emphasis has been placed on the synthesis strategies, structural design, properties and possible mechanisms that are perhaps responsible for their improved theranostic characteristics. Finally, we discuss the critical issues that may accelerate the development of carbon-based nanostructured materials for application in cancer theranostics.