Ferrocene or ferrocenium has been widely studied in the field of organometallic complexes because of its stable thermodynamic, kinetic and redox properties. Novel hexaferrocenium tri[hexa(isothiocyanato)iron(III)]trihydroxonium (HexaFc) complex was the product from the reaction of ferrocene, maleic acid and ammonium thiocyanate and was confirmed by elemental analysis CHNS, FTIR and single crystal X-ray crystallography. In this study, HexaFc was used for the first time as an electroactive indicator for porcine DNA biosensor. The UV-Vis DNA titrations with this compound showed hypochromism and redshift at 250 nm with increasing DNA concentrations. The binding constant (Kb) for HexaFc complex towards CT-DNA (calf-thymus DNA) was 3.1 × 104 M-1, indicated intercalator behaviour of the complex. To test the usefulness of this complex for DNA biosensor application, a porcine DNA biosensor was constructed. The recognition probes were covalently immobilised onto silica nanospheres (SiNSs) via glutaraldehyde linker on a screen-printed electrode (SPE). After intercalation with the HexaFc complex, the response of the biosensor to the complementary porcine DNA was measured using differential pulse voltammetry. The DNA biosensor demonstrated a linear response range to the complementary porcine DNA from 1 × 10-6 to 1 × 10-3 µM (R2 = 0.9642) with a limit detection of 4.83 × 10-8 µM and the response was stable up to 23 days of storage at 4 °C with 86% of its initial response. The results indicated that HexaFc complex is a feasible indicator for the DNA hybridisation without the use of a chemical label for the detection of porcine DNA.
Coronavirus disease 2019 (COVID-19) is a highly contagious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Diagnosis of COVID-19 depends on quantitative reverse transcription PCR (qRT-PCR), which is time-consuming and requires expensive instrumentation. Here, we report an ultrasensitive electrochemical biosensor based on isothermal rolling circle amplification (RCA) for rapid detection of SARS-CoV-2. The assay involves the hybridization of the RCA amplicons with probes that were functionalized with redox active labels that are detectable by an electrochemical biosensor. The one-step sandwich hybridization assay could detect as low as 1 copy/μL of N and S genes, in less than 2 h. Sensor evaluation with 106 clinical samples, including 41 SARS-CoV-2 positive and 9 samples positive for other respiratory viruses, gave a 100% concordance result with qRT-PCR, with complete correlation between the biosensor current signals and quantitation cycle (Cq) values. In summary, this biosensor could be used as an on-site, real-time diagnostic test for COVID-19.
Designing a biosensor for versatile biomedical applications is a sophisticated task and how dedicatedly functionalized fullerene (C60) can perform on this stage is a challenge for today and tomorrow's nanoscience and nanotechnology. Since the invention of biosensor, many ideas and methods have been invested to upgrade the functionality of biosensors. Due to special physicochemical characteristics, the novel carbon material "fullerene" adds a new dimension to the construction of highly sensitive biosensors. The prominent aspects of fullerene explain its outstanding performance in biosensing devices as a mediator, e.g. fullerene in organic solvents exhibits five stages of reversible oxidation/reduction, and hence fullerene can work either as an electrophile or nucleophile. Fullerene is stable and its spherical structure produces an angle strain which allows it to undergo characteristic reactions of addition to double bonds (hybridization which turns from sp(2) to sp(3)). Research activities are being conducted worldwide to invent a variety of methods of fullerene functionalization with a purpose of incorporating it effectively in biosensor devices. The different types of functionalization methods include modification of fullerene into water soluble derivatives and conjugation with enzymes and/or other biomolecules, e.g. urease, glucose oxidase, hemoglobin, myoglobin (Mb), conjugation with metals e.g. gold (Au), chitosan (CS), ferrocene (Fc), etc. to enhance the sensitivity of biosensors. The state-of-the-art research on fullerene functionalization and its application in sensor devices has proven that fullerene can be implemented successfully in preparing biosensors to detect glucose level in blood serum, urea level in urine solution, hemoglobin, immunoglobulin, glutathione in real sample for pathological purpose, to identify doping abuse, to analyze pharmaceutical preparation and even to detect cancer and tumor cells at an earlier stage. Employing fullerene-metal matrix for the detection of tumor and cancer cells is also possible by the inclusion of fullerene in single-walled carbon nanotubes (SWCNTs) known as peapods as well as in double-walled carbon nanotubes (DWCNTs), to augment the effectiveness of biosensors. This review discusses various approaches that have been reported for functionalizing fullerene (C60) derivatives and their application in different types of biosensor fabrication.
A new alcohol oxidase (AOX) enzyme-based formaldehyde biosensor based on acrylic microspheres has been developed. Hydrophobic poly(n-butyl acrylate-N-acryloxy-succinimide) [poly(nBA-NAS)] microspheres, an enzyme immobilization matrix, was synthesized using photopolymerization in an emulsion form. AOX-poly(nBA-NAS) microspheres were deposited on a pH transducer made from a layer of photocured and self-plasticized polyacrylate membrane with an entrapped pH ionophore coated on a Ag/AgCl screen printed electrode (SPE). Oxidation of formaldehyde by the immobilized AOX resulted in the production of protons, which can be determined via the pH transducer. Effects of buffer concentrations, pH and different amount of immobilization matrix towards the biosensor's analytical performance were investigated. The formaldehyde biosensor exhibited a dynamic linear response range to formaldehyde from 0.3-316.2 mM and a sensitivity of 59.41 ± 0.66 mV/decade (R(2) = 0.9776, n = 3). The lower detection limit of the biosensor was 0.3 mM, while reproducibility and repeatability were 3.16% RSD (relative standard deviation) and 1.11% RSD, respectively (n = 3). The use of acrylic microspheres in the potentiometric formaldehyde biosensor improved the biosensor's performance in terms of response time, linear response range and long term stability when compared with thick film immobilization methods.
A single-step fabrication of a glucose biosensor with simultaneous immobilization of both ferrocene mediator and glucose oxidase in a photocurable methacrylic film consisting of poly(methyl methacrylate-co-2-hydroxylethyl methacrylate) was reported. The entrapped ferrocene showed reversible redox behaviour in the photocured film and no significant leaching of both entrapped ferrocene and enzyme glucose oxidase was observed because of the low water absorption properties of the co-polymer films. From electrochemical studies, ferrocene entrapped in the co-polymer film demonstrated slow diffusion properties. A linear glucose response range of 2-11 mM was obtained at low applied potential of +0.25 V. The glucose biosensor fabricated by this photocuring method yielded sensor reproducibility and repeatability with relative standard deviation of <10% and long-term stability of up to 14 days. The main advantage of the use of photocurable procedure is that biosensor membrane fabrication can be performed in a single step without any lengthy chemical immobilization of enzyme.
The fabrication of an electrochemical sensor based on an iron oxide/graphene modified glassy carbon electrode (Fe3O4/rGO/GCE) and its simultaneous detection of dopamine (DA) and ascorbic acid (AA) is described here. The Fe3O4/rGO nanocomposite was synthesized via a simple, one step in-situ wet chemical method and characterized by different techniques. The presence of Fe3O4 nanoparticles on the surface of rGO sheets was confirmed by FESEM and TEM images. The electrochemical behavior of Fe3O4/rGO/GCE towards electrocatalytic oxidation of DA was investigated by cyclic voltammetry (CV) and differential pulse voltammetry (DPV) analysis. The electrochemical studies revealed that the Fe3O4/rGO/GCE dramatically increased the current response against the DA, due to the synergistic effect emerged between Fe3O4 and rGO. This implies that Fe3O4/rGO/GCE could exhibit excellent electrocatalytic activity and remarkable electron transfer kinetics towards the oxidation of DA. Moreover, the modified sensor electrode portrayed sensitivity and selectivity for simultaneous determination of AA and DA. The observed DPVs response linearly depends on AA and DA concentration in the range of 1-9 mM and 0.5-100 µM, with correlation coefficients of 0.995 and 0.996, respectively. The detection limit of (S/N = 3) was found to be 0.42 and 0.12 µM for AA and DA, respectively.
Alternative sensory systems for the development of prosthetic knees are being increasingly highlighted nowadays, due to the rapid advancements in the field of lower limb prosthetics. This study presents the use of piezoelectric bimorphs as in-socket sensors for transfemoral amputees. An Instron machine was used in the calibration procedure and the corresponding output data were further analyzed to determine the static and dynamic characteristics of the piezoelectric bimorph. The piezoelectric bimorph showed appropriate static operating range, repeatability, hysteresis, and frequency response for application in lower prosthesis, with a force range of 0-100 N. To further validate this finding, an experiment was conducted with a single transfemoral amputee subject to measure the stump/socket pressure using the piezoelectric bimorph embedded inside the socket. The results showed that a maximum interface pressure of about 27 kPa occurred at the anterior proximal site compared to the anterior distal and posterior sites, consistent with values published in other studies. This paper highlighted the capacity of piezoelectric bimorphs to perform as in-socket sensors for transfemoral amputees. However, further experiments are recommended to be conducted with different amputees with different socket types.
A fluorescence-based fiber optic toxicity biosensor based on genetically modified Escherichia coli (E. coli) with green fluorescent protein (GFP) was developed for the evaluation of the toxicity of several hazardous heavy metal ions. The toxic metals include Cu(II), Cd(II), Pb(II), Zn(II), Cr(VI), Co(II), Ni(II), Ag(I) and Fe(III). The optimum fluorescence excitation and emission wavelengths of the optical biosensor were 400 ± 2 nm and 485 ± 2 nm, respectively. Based on the toxicity observed under optimal conditions, the detection limits of Cu(II), Cd(II), Pb(II), Zn(II), Cr(VI), Co(II), Ni(II), Ag(I) and Fe(III) that can be detected using the toxicity biosensor were at 0.04, 0.32, 0.46, 2.80, 100, 250, 400, 720 and 2600 μg/L, respectively. The repeatability and reproducibility of the proposed biosensor were 3.5%-4.8% RSD (relative standard deviation) and 3.6%-5.1% RSD (n = 8), respectively. The biosensor response was stable for at least five weeks, and demonstrated higher sensitivity towards metal toxicity evaluation when compared to a conventional Microtox assay.
Reporting biomolecular interactions has become part and parcel of many applications of science towards an in-depth understanding of disease and gene regulation. Apart from that, in diagnostic applications where biomolecules (antibodies and aptamers) are vastly applied, meticulous monitoring of biomolecular interaction is vital for clear-cut diagnosis. Several currently available methods of analyzing the interaction of the ligands with the appropriate analytes are aided by labeling using fluorescence or luminescence techniques. However, labeling is cumbersome and can occupy important binding sites of interactive molecules to be labeled, which may interfere with the conformational changes of the molecules and increase non-specificity. Optical-based sensing can provide an alternative way as a label-free procedure for monitoring biomolecular interactions. Optical sensors affiliated with different operating principles, including surface plasmon changes, scattering and interferometry, can impart a huge impact for in-house and point-of-care applications. This optical-based biosensing permits real-time monitoring, obviating the use of hazardous labeling molecules such as radioactive tags. Herein, label-free ways of reporting biomolecular interactions by various optical biosensors were gleaned.
Reduced graphene oxide (rGO) has emerged as a promising nanomaterial for reliable detection of pathogenic bacteria due to its exceptional properties such as ultrahigh electron transfer ability, large surface to volume ratio, biocompatibility, and its unique interactions with DNA bases of the aptamer. In this study, rGO-azophloxine (AP) nanocomposite aptasensor was developed for a sensitive, rapid, and robust detection of foodborne pathogens. Besides providing an excellent conductive and soluble rGO nanocomposite, the AP dye also acts as an electroactive indicator for redox reactions. The interaction of the label-free single-stranded deoxyribonucleic acid (ssDNA) aptamer with the test organism, Salmonella enterica serovar Typhimurium (S. Typhimurium), was monitored by differential pulse voltammetry analysis, and this aptasensor showed high sensitivity and selectivity for whole-cell bacteria detection. Under optimum conditions, this aptasensor exhibited a linear range of detection from 108 to 101 cfu mL-1 with good linearity (R 2 = 0.98) and a detection limit of 101 cfu mL-1. Furthermore, the developed aptasensor was evaluated with non-Salmonella bacteria and artificially spiked chicken food sample with S. Typhimurium. The results demonstrated that the rGO-AP aptasensor possesses high potential to be adapted for the effective and rapid detection of a specific foodborne pathogen by an electrochemical approach. Graphical abstract Fabrication of graphene-based nanocomposite aptasensor for detection of foodborne pathogen.
A novel third generation H2O2 biosensor is fabricated using multiporous SnO2 nanofiber/carbon nanotubes (CNTs) composite as a matrix for the immobilization of redox protein onto glassy carbon electrode. The multiporous nanofiber (MPNFs) of SnO2 is synthesized by electrospinning technique from the tin precursor. This nanofiber shows high surface area and good electrical conductivity. The SnO2 nanofiber/CNT composite increases the efficiency of biomolecule loading due to its high surface area. The morphology of the nanofiber has been evaluated by scanning electron microscopy (SEM). Cyclic Voltammetry and amperometry technique are employed to study and optimize the performance of the fabricated electrode. A direct electron transfer between the protein's redox centre and the glassy carbon electrode is established after fabrication of the electrode. The fabricated electrode shows excellent electrocatalytic reduction to H2O2. The catalysis currents increases linearly to the H2O2 concentration in a wide range of 1.0 10-6-1.4×10-4M and the lowest detection limit was 30nM (S/N=3). Moreover, the biosensor showed a rapid response to H2O2, a good stability and reproducibility.
A novel nitrogen/argon (N2/Ar) radio frequency (RF) plasma functionalized graphene nanosheet/graphene nanoribbon (GS/GNR) hybrid material (N2/Ar/GS/GNR) was developed for simultaneous determination of ascorbic acid (AA), dopamine (DA) and uric acid (UA). Various nitrogen mites introduced into GS/GNR hybrid structure was evidenced by a detailed microscopic, spectroscopic and surface area analysis. Owing to the unique structure and properties originating from the enhanced surface area, nitrogen functional groups and defects introduced on both the basal and edges, N2/Ar/GS/GNR/GCE showed high electrocatalytic activity for the electrochemical oxidations of AA, DA, and UA with the respective lowest detection limits of 5.3, 2.5 and 5.7 nM and peak-to-peak separation potential (ΔEP) (vs Ag/AgCl) in DPV of 220, 152 and 372 mV for AA/DA, DA/UA and AA/UA respectively. Moreover, the selectivity, stability, repeatability and excellent performance in real time application of the fabricated N2/Ar/GS/GNR/GCE electrode suggests that it can be considered as a potential electrode material for simultaneous detection of AA, DA, and UA.
Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, significantly affecting the rate of human mortality and morbidity. Conventional methods for the detection of V. parahaemolyticus such as culture-based methods, immunological assays, and molecular-based methods require complicated sample handling and are time-consuming, tedious, and costly. Recently, biosensors have proven to be a promising and comprehensive detection method with the advantages of fast detection, cost-effectiveness, and practicality. This research focuses on developing a rapid method of detecting V. parahaemolyticus with high selectivity and sensitivity using the principles of DNA hybridization. In the work, characterization of synthesized polylactic acid-stabilized gold nanoparticles (PLA-AuNPs) was achieved using X-ray Diffraction (XRD), Ultraviolet-visible Spectroscopy (UV-Vis), Transmission Electron Microscopy (TEM), Field-emission Scanning Electron Microscopy (FESEM), and Cyclic Voltammetry (CV). We also carried out further testing of stability, sensitivity, and reproducibility of the PLA-AuNPs. We found that the PLA-AuNPs formed a sound structure of stabilized nanoparticles in aqueous solution. We also observed that the sensitivity improved as a result of the smaller charge transfer resistance (Rct) value and an increase of active surface area (0.41 cm2). The development of our DNA biosensor was based on modification of a screen-printed carbon electrode (SPCE) with PLA-AuNPs and using methylene blue (MB) as the redox indicator. We assessed the immobilization and hybridization events by differential pulse voltammetry (DPV). We found that complementary, non-complementary, and mismatched oligonucleotides were specifically distinguished by the fabricated biosensor. It also showed reliably sensitive detection in cross-reactivity studies against various food-borne pathogens and in the identification of V. parahaemolyticus in fresh cockles.
Human papillomavirus (HPV) is one of the most common sexually transmitted disease, transmitted through intimate skin contact or mucosal membrane. The HPV virus consists of a double-stranded circular DNA and the role of HPV virus in cervical cancer has been studied extensively. Thus it is critical to develop rapid identification method for early detection of the virus. A portable biosensing device could give rapid and reliable results for the identification and quantitative determination of the virus. The fabrication of electrochemical biosensors is one of the current techniques utilized to achieve this aim. In such electrochemical biosensors, a single-strand DNA is immobilized onto an electrically conducting surface and the changes in electrical parameters due to the hybridization on the electrode surface are measured. This review covers the recent developments in electrochemical DNA biosensors for the detection of HPV virus. Due to the several advantages of electrochemical DNA biosensors, their applications have witnessed an increased interest and research focus nowadays.
The simultaneous measurement of the concentration of anticancer drugs with a fast, sensitive and accurate method in biological samples is a challenge for better monitoring of drug therapy and better determine the pharmacokinetics. An electrochemical sensor was developed for the simultaneous determination of anticancer drugs, Ifosfamide (IFO) and Etoposide (ETO) based on pencil graphite electrode modified with Au/Pd@rGO nanocomposite decorated with poly (L-Cysteine). Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were utilized to study the properties of the modified electrode. The electrochemical behavior of IFO and ETO on the Au/Pd@rGO@p(L-Cys) modified electrode was investigated by cyclic voltammetry and differential pulse voltammetry (DPV) techniques and the obtained results confirmed its efficiency for the individual and simultaneous sensing of IFO and ETO. After optimization of electrochemical parameters, the fabricated sensor presented excellent performance in simultaneous determination of IFO and ETO with a wide linear range from 0.10 to 90.0 μM and 0.01 to 40.0 μM and low detection limits (3 Sb/m) of 9.210 nM and 0.718 nM, respectively. In addition, this study proved that the constructed sensor could be useful to simultaneous analysis of IFO and ETO in biological samples and pharmaceutical compounds.
The inherent ability of nucleic acids to recognize a complementary pair has gained wide popularity in DNA sensor applications. DNA molecules can be produced in bulk and easily incorporated with various nanomaterials for sensing applications. More complex designs and sophisticated DNA sensors have been reported over the years to allow DNA detection in a faster, cheaper, and more convenient manner. Here, we report a DNA sensor designed to function like a switch to turn "on" silver nanocluster (AgNC) generation in the presence of a specific DNA target. By defining the probe region sequence, we are able to tune the color of the AgNC generated in direct relation to the different targets. As a proof of concept, we used dengue RNA-dependent RNA polymerase conserved sequences from all four serotypes as targets. This method was able to distinguish each dengue serotype by generating the serotype-respective AgNCs. The DNA switch was also able to identify and amplify the correct target in a mixture of targets with good specificity. This strategy has a detection limit of between 1.5 and 2.0 µM depending on the sequence of AgNC. The DNA switch approach provides an attractive alternative for single-target or multiplex DNA detection.
Interaction between split RNA aptamer and the clinically important target, HIV-1 Tat was investigated on a biosensing surface transduced by functionally choreographed multiwall carbon nanotubes (MWCNTs). Acid oxidation was performed to functionalize MWCNTs with carboxyl functional groups. X-ray photoelectron spectroscopy analysis had profound ~2.91% increment in overall oxygen group and ~1% increment was noticed with a specific carboxyl content owing to CO and OCO bonding. The interaction between split RNA aptamer and HIV-1 Tat protein was quantified by electrical measurements with the current signal (Ids) over a gate voltage (Vgs). Initially, 34.4 mV gate voltage shift was observed by the immobilization of aptamer on MWCNT. With aptamer and HIV-1 Tat interaction, the current flow was decreased with the concomitant gate voltage shift of 23.5 mV. The attainment of sensitivity with split aptamer and HIV-1 Tat interaction on the fabricated device was 600 pM. To ensure the genuine interaction of aptamer with HIV-1 Tat, other HIV-1 proteins, Nef and p24 were interacted with aptamer and they displayed the negligible interferences with gate voltage shift of 3.5 mV and 5.7 mV, which shows 4 and 2.5 folds lesser than HIV-1 Tat interaction, respectively.
A fluorogenic probe has been developed for determination of telomerase activity using chimeric DNA-templated silver nanoclusters (AgNCs). The formation of AgNCs was investigated before (route A) and after (route B) telomerase elongation reaction. Both routes caused selective quenching of the yellow emission of the AgNCs (best measured at excitation/emission wavelength of 470/557 nm) in telomerase-positive samples. The quenching mechanism was studied using synthetically elongated DNA to mimic the telomerase-catalyzed elongation. The findings show that quenching is due to the formation of parallel G-quadruplexes with a -TTA- loop in the telomerase elongated products. The assay was validated using different cancer cell extracts, with intra- and interassay coefficients of variations of <9.8%. The limits of detection for MCF7, RPMI 2650 and HT29 cell lines are 15, 22 and 39 cells/μL. This represents a distinct improvement over the existing telomeric repeat amplification protocol (TRAP) assay in terms of time, sensitivity and cost. Graphical Abstract A method was developed using chimeric DNA-templated silver nanoclusters to detect telomerase activity directly in cell extracts. The sensitivity of this new method outperforms the traditional TRAP assay, and without the need for amplification.
This study highlighted the performance of the developed integrated loop-mediated isothermal amplification (LAMP) coupled with a colorimetric DNA-based magnetogenosensor. The biosensor operates through a DNA hybridization system in which a specific designed probe captures the target LAMP amplicons. We demonstrated the magnetogenosensor assay by detecting pathogenic Leptospira, which causes leptospirosis. The color change of the assay from brown to blue indicated a positive result, whereas a negative result was indicated by the assay maintaining its brown color. The DNA biosensor was able to detect DNA at a concentration as low as 200 fg/μl, which is equivalent to 80 genomes/reaction. The specificity of the biosensor assay was 100% when it was evaluated with 172 bacterial strains. An integrated LAMP and probe-specific magnetogenosensor was successfully developed, promising simple and rapid visual detection in clinical diagnostics and service as a point-of-care device.
Two-dimensional (2D) layered nanomaterials have triggered an intensive interest due to the fascinating physiochemical properties with the exceptional physical, optical and electrical characteristics that transpired from the quantum size effect of their ultra-thin structure. Among the family of 2D nanomaterials, molybdenum disulfide (MoS2) features distinct characteristics related to the existence of direct energy bandgap, which significantly lowers the leakage current and surpasses other 2D materials. In this overview, we expatiate the novel strategies to synthesize MoS2 that cover techniques such as liquid exfoliation, chemical vapour deposition, mechanical exfoliation, hydrothermal reaction, and Van Der Waal epitaxial growth on the substrate. We extend the discussion on the recent progress in biosensing applications of the produced MoS2, highlighting the important surface-to-volume of ultrathin MoS2 structure, which enhances the overall performance of the devices. Further, envisioned the missing piece with the current MoS2-based biosensors towards developing the future strategies.