The envelope glycoprotein domain III (EDIII) of dengue virus (DENV) has been recognised as the antigenic region responsible for receptor binding. In the present work, we have proposed a novel immunosensor constructed on a graphene-coated screen-printed carbon electrode (SPCE) using plant-derived EDIII as the probe antigen to target DENV IgG antibodies. The developed immunosensor demonstrated high sensitivity towards DENV IgG within a wide linear working range (125-2000 ng mL-1) under the optimised sensing conditions. The limit of detection was determined to be 22.5 ng mL-1. The immunosensor also showed high specificity towards DENV IgG, capable of differentiating DENV IgG from the antibodies of other infectious diseases including the similarly structured Zika virus (ZIKV). The ability of the immunosensor to detect dengue antibodies in serum samples was also verified by conducting tests on mouse serum samples. The proposed immunosensor was able to provide a binary (positive/negative) response towards the serum samples comparable to the conventional enzyme-linked immunosorbent assay (ELISA), indicating promising potential for realistic applications.
With the long-term goal of developing an ultra-sensitive microcantilever-based biosensor for versatile biomarker detection, new controlled bioreceptor-analytes systems are being explored to overcome the disadvantages of conventional ones. Gold (Au) microwires have been used as a probe to overcome the tolerance problem that occurs in response to changes in environmental conditions. However, the cytotoxicity of Au microwires is still unclear. Here, we examined the cytotoxicity of Au microwires systems using both commercial and as-synthesised Au microwires. In vitro experiments show that commercial Au microwires with an average quoted length of 5.6 µm are highly toxic against Gram-negative Escherichia coli (E. coli) at 50 µg/mL. However, this toxicity is due to the presence of CTAB surfactant not by the microwires. Conversely, the as-synthesised Au microwires show non-cytotoxicity even at the maximum viable concentration (330 µg/mL). These findings may lead to the development of potentially life-saving cytotoxicity-free biosensors for an early diagnostic of potential diseases.
Aptamers are short single-stranded oligonucleotides (either DNA or RNA) that can fold into well-defined three-dimensional (3D) spatial structures which enable them to capture their specific target by complementary shape interactions. Aptamers are selected from large random libraries through the SELEX process and only a small fraction of the sequence is involved in direct docking with the target. In this paper, we describe the possible truncation variants of zearalenone (ZEA) aptamer which might be an effective binding region for the target. The originally selected zearalenone (ZEA) aptamer was 80-mer in length and shown to bind the target with a high affinity (Kd = 41 ± 5 nM). Herein, computational docking simulation was performed with 15 truncated variants to determine the predicted binding energy and responsible binding site of the aptamer-analyte complex. The results revealed that 5 truncated variants had binding energy lower than - 7.0 kcal/mol. Circular dichroism analysis was performed on the shortlisted aptamer and the conformational change of aptamers was observed with the presence of an analyte. Aptamer Z3IN (29-mer) was chosen as the most enhanced affinity for its target with a dissociation constant of 11.77 ± 1.44 nM. The aptamer was further applied in the electrochemical aptasensor of ZEA based on an indirect competitive format. The results demonstrated that the truncated aptamer leads to an enhancement of the sensitivity of the biosensor.
The presence of heavy metal in food chains due to the rapid industrialization poses a serious threat on the environment. Therefore, detection and monitoring of heavy metals contamination are gaining more attention nowadays. However, the current analytical methods (based on spectroscopy) for the detection of heavy metal contamination are often very expensive, tedious and can only be handled by trained personnel. DNA biosensors, which are based on electrochemical transduction, is a sensitive but inexpensive method of detection. The principles, sensitivity, selectivity and challenges of electrochemical biosensors are discussed in this review. This review also highlights the major advances of DNA-based electrochemical biosensors for the detection of heavy metal ions such as Hg(2+), Ag(+), Cu(2+) and Pb(2+).
This study highlighted the development of a four target nitrocellulose-based nucleic acid lateral flow immunoassay biosensor in a dry-reagent strip format for interpretation of double-labelled double-stranded amplicons from thermostabilised triplex loop-mediated isothermal amplification assay. The DNA biosensor contained two test lines which captured biotin and texas red labelled amplicons; a LAMP internal amplification control line that captured digoxigenin labelled amplicon; and a chromatography control line that validated the functionality of the conjugated gold nanoparticles and membrane. The red lines on detection pad were generated when the gold nanoparticles conjugated antibody bound to the fluorescein labelled amplicons, and the capture agents bound to their specific hapten on the other 5' end of the double-stranded amplicon. The applicability of this DNA biosensor was demonstrated using amoebiasis-causing Entamoeba histolytica simultaneously with the non-pathogenic but morphologically identical Entamoeba dispar and Entamoeba moshkovskii. The biosensor detection limit was 10 E. histolytica trophozoites, and revealed 100% specificity when it was evaluated against 3 medically important Entamoeba species and 75 other pathogenic microorganisms. Heat stability test showed that the biosensor was stable for at least 181 days at ambient temperature. This ready-to-use and cold-chain-free biosensor facilitated the post-LAMP analysis based on visualisation of lines on strip instead of observation of amplicon patterns in agarose gel.
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 miniaturized biosensing platform, based on monoclonal amyloid-beta antibodies (mAβab) that were immobilized on a disc-shaped platinum/iridium (Pt/Ir) microelectrode surface coupled with an impedimetric signal transducer, was developed for the label-free and sensitive detection of amyloid-beta peptide fragment 1-40 (Aβ40); a reliable biomarker for early diagnosis of Alzheimer's disease (AD). A Pt/Ir microelectrode was electropolymerized with poly (ortho-phenylenediamine), a conducting free amine-containing aromatic polymer; followed by crosslinking with glutaraldehyde for subsequent coupling of mAβab on the microelectrode surface. This modification strategy efficiently improved the impedimetric detection performance of Aβ40 in terms of charge transfer resistance (∼400-fold difference) and normalized impedance magnitude percentage change (∼40% increase) compared with a passive adsorption-based immobilization method. The sensitivity of the micro-immunosensing assay was found to be 1056 kΩ/(pg/mL)/cm2 and the limit of detection was found to be 4.81 pg/mL with a dynamic range of 1-104 pg/mL (R2 = 0.9932). The overall precision of the assay, as measured by relative standard deviation, ranged from 0.84 to 5.15%, demonstrating its reliability and accuracy; while in respect to assay durability and stability, the immobilized mAβab were able to maintain 80% of their binding activity to Aβ40 after incubation for 48 h at ambient temperature (25 °C). To validate the practical applicability, the assay was tested using brain tissue lysates prepared from AD-induced rats. Results indicate that the proposed impedimetric micro-immunosensing platform is highly versatile and adaptable for the quantitative detection of other disease-related biomarkers.
A method to fabricate a bioinspired nanobiosensor using electronic-based artificial taste receptors for glucose diagnosis is presented. Fabricated bioinspired glucose nanobiosensor designated based on an artificial taste bud including an amperometric glucose biosensor and taste bud-inspired circuits. In fact, the design of the taste bud-inspired circuits was inspired by the signal-processing mechanism of taste nerves which involves two layers. The first, known as a type II cell, detects the glucose by glucose oxidase and transduces the current signal obtained for the pulse pattern is conducted to the second layer, called type III cell, to induce synchronisation of the neural spiking activity. The oscillation results of fabricated bioinspired glucose nanobiosensor confirmed an increase in the frequency of the output pulse as a function of the glucose concentration. At high glucose concentrations, the bioinspired glucose nanobiosensor showed a pulse train of alternating short and long interpulse intervals. A computational analysis performed to validate the hypothesis, which was successfully reproduced the alternating behaviour of bioinspired glucose our nanobiosensor by increasing the output frequency and alternation of pulse intervals according to the reduction in the resistivity of the biosensor.
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 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.
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.
Light-addressable potentiometric sensors (LAPS) are of great interest in bioimaging applications such as the monitoring of concentrations in microfluidic channels or the investigation of metabolic and signaling events in living cells. By measuring the photocurrents at electrolyte-insulator-semiconductor (EIS) and electrolyte-semiconductor structures, LAPS can produce spatiotemporal images of chemical or biological analytes, electrical potentials and impedance. However, its commercial applications are often restricted by their limited AC photocurrents and resolution of LAPS images. Herein, for the first time, the use of 1D semiconducting oxides in the form of ZnO nanorods for LAPS imaging is explored to solve this issue. A significantly increased AC photocurrent with enhanced image resolution has been achieved based on ZnO nanorods, with a photocurrent of 45.7 ± 0.1 nA at a light intensity of 0.05 mW, a lateral resolution as low as 3.0 μm as demonstrated by images of a PMMA dot on ZnO nanorods and a pH sensitivity of 53 mV/pH. The suitability of the device for bioanalysis and bioimaging was demonstrated by monitoring the degradation of a thin poly(ester amide) film with the enzyme α-chymotrypsin using LAPS. This simple and robust route to fabricate LAPS substrates with excellent performance would provide tremendous opportunities for bioimaging.
Highly oriented ZnO nanorod (NR) arrays were fabricated on a seeded substrate through a hydrothermal route. The prepared ZnO nanorods were used as an amperometric enzyme electrode, in which glucose oxidase (GOx) was immobilised through physical adsorption. The modified electrode was designated as Nafion/GOx/ZnO NRs/ITO. The morphology and structural properties of the fabricated ZnO nanorods were analysed using field-emission scanning electron microscope and X-ray diffractometer. The electrochemical properties of the fabricated biosensor were studied by cyclic voltammetry and amperometry. Electrolyte pH, electrolyte temperature and enzyme concentration used for immobilisation were the examined parameters influencing enzyme activity and biosensor performance. The immobilised enzyme electrode showed good GOx retention activity. The amount of electroactive GOx was 7.82 × 10-8 mol/cm2, which was relatively higher than previously reported values. The Nafion/GOx/ZnO NRs/ITO electrode also displayed a linear response to glucose ranging from 0.05 mM to 1 mM, with a sensitivity of 48.75 µA/mM and a low Michaelis-Menten constant of 0.34 mM. Thus, the modified electrode can be used as a highly sensitive third-generation glucose biosensor with high resistance against interfering species, such as ascorbic acid, uric acid and L-cysteine. The applicability of the modified electrode was tested using human blood samples. Results were comparable with those obtained using a standard glucometer, indicating the excellent performance of the modified electrode.
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.
Surface enhanced Raman scattering (SERS) DNA biosensing is an ultrasensitive, selective, and rapid detection technique with the ability to produce molecule-specific distinct fingerprint spectra. It supersedes the long amplicon based PCR assays, the fluorescence and spectroscopic techniques with their quenching and narrow spectral bandwidth, and the electrochemical detection techniques using multiplexing. However, the performance of the SERS DNA biosensor relies on the DNA probe length, platform composition, both the presence and position of Raman tags and the chosen sensing strategy. In this context, we herein report a SERS biosensor based on dual nanoplatforms with a uniquely designed Raman tag (ATTO Rho6G) intercalated short-length DNA probe for the sensitive detection of the pig species Sus scrofa. In the design of the signal probe (SP), a Raman tag was incorporated adjacent to the spacer arm, followed by a terminal thiol modifier, which consequently had a strong influence on the SERS signal enhancement. The detection strategy involves the probe-target DNA hybridization mediated coupling of the two platforms, i.e., the graphene oxide-gold nanorod (GO-AuNR) functionalized capture probe (CP) and SP-conjugated gold nanoparticles (AuNPs), consequently enhancing the SERS intensity by both the electromagnetic hot spots generated at the junctions or interstices of the two platforms and the chemical enhancement between the AuNPs and the adsorbed intercalated Raman tag. This dual platform based SERS DNA biosensor exhibited outstanding sensitivity in detecting pork DNA with a limit of detection (LOD) of 100 aM validated with DNA extracted from a pork sample (LOD 1 fM). Moreover, the fabricated SERS biosensor showed outstanding selectivity and specificity for differentiating the DNA sequences of six closely related non-target species from the target DNA sequences with single and three nucleotide base-mismatches. Therefore, the developed short-length DNA linked dual platform based SERS biosensor could replace the less sensitive traditional methods of pork DNA detection and be adopted as a universal detection approach for the qualitative and quantitative detection of DNA from any source.
Toxicity Identification Evaluation (TIE) is a useful method for the classification and identification of toxicants in a composite environment water sample. However, its extension to a larger sample size has been restrained owing to the limited throughput of toxicity bioassays. Here we reported the development of a high-throughput method of TIE Phase I. This newly developed method was assisted by the fluorescence-based cellular oxidation (CO) biosensor fabricated with roGFP2-expressing bacterial cells in 96-well microplate format. The assessment of four river water samples from Langat river basin by this new method demonstrated that the contaminant composition of the four samples can be classified into two distinct groups. The entire toxicity assay consisted of 2338 tests was completed within 12 h with a fluorescence microplate reader. Concurrently, the sample volume for each assay was reduced to 50 μL, which is 600 to 4700 times lesser to compare with conventional bioassays. These imply that the throughput of the CO biosensor-assisted TIE Phase I is now feasible for constructing a large-scale toxicity monitoring system, which would cover a whole watershed scale.
Novel biosensor architecture based on nanocrystalline cellulose (NCC)/CdS quantum dots (QDs) nanocomposite was developed for phenol determination. This nanocomposite was prepared with slight modification of nanocrystalline cellulose (NCC) with cationic surfactant of cetyltriammonium bromide (CTAB) and further decorated with 3-mercaptopropionic acid (3-MPA) capped CdS QDs. The nanocomposite material was then employed as scaffold for immobilization of tyrosinase enzyme (Tyr). The electrocatalytic response of Tyr/CTAB-NCC/QDs nanocomposite towards phenol was evaluated using differential pulse voltammetry (DPV). The current response obtained is proportional to the concentration of phenol which attributed to the reduction of o-quinone produced at the surface of the modified electrode. Under the optimal conditions, the biosensor exhibits good linearity towards phenol in the concentration range of 5-40 μM (R2 = 0.9904) with sensitivity and limit of detection (LOD) of 0.078 μA/μM and 0.082 μM, respectively.
This paper primarily demonstrates the approach to enhance the sensing performance on antigen C-reactive protein (CRP) and anti-CRP antibody binding event. A nanogapped electrode structure with the gap of ~100 nm was modified by the anti-CRP antibody (Probe) to capture the available CRP. In order to increase the amount of antigen to be captured, a gold nanorod with 119 nm in length and 25 nm in width was integrated, to increase the surface area. A comparative study between the existence and non-existence of gold nanorod utilization was evaluated. Analysis of the sensing surface was well-supported by atomic force microscopy, scanning electron microscopy, 3D nano-profilometry, high-power microscopy and UV-Vis spectroscopy. The dielectric voltammetric analysis was carried out from 0 V to 2 V. The sensitivity was calculated based on 3σ and attained as low as 1 pM, which is tremendously low compared to real CRP concentration (119 nM) in human blood serum. The gold nanorod conjugation with antibody has enhanced the sensitivity to 100 folds (10 fM). The specificity of the CRP detection by the proposed strategy was anchored by ELISA and failure in the detection of human blood clotting factor IX by voltammetry. Despite, CRP antigen was further detected in human serum by spiking CRP to run-through the detection with the physiologically relevant samples.
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.
Proteases are key signalling molecules for many physiological processes and their dysregulation is implicated in the progression of a range of diseases. Sensitive methods to measure protease activities in complex biological samples are critical for rapid disease diagnoses. The proteolytic activity of plasmin reflects the fibrinolysis state of blood and its deregulation can indicate pathologies such as bleeding events. While Bioluminescence Resonance Energy Transfer (BRET) is a powerful and sensitive method for the detection of protease activity, the commonly applied blue-shifted BRET2 system, consisting of the Renilla luciferase Rluc2 and the large-stokes shift fluorescent protein GFP2, suffers from light absorption and light scattering in human plasma samples. To address this challenge, we developed a red-shifted BRET-based plasmin sensor by substituting BRET2 with the BRET6 system. BRET6 is composed of the red-shifted RLuc8.6 luciferase linked to the red light emitting fluorescent protein TurboFP635. The BRET6 biosensor exhibited 3-fold less light absorption in plasma samples compared to the BRET2 sensor leading to an up to a 5-fold increase in sensitivity for plasmin detection in plasma. The limits of detection for plasmin were determined to be 11.90 nM in 7.5% (v/v) plasma with a 10 min assay which enables biologically relevant plasmin activities of thrombolytic therapies to be detected. While a colorigenic plasmin activity assay achieved a similar detection limit of 10.91 nM in 7.5% (v/v) human plasma, it required a 2 h incubation period. The BRET6 sensor described here is faster and more specific than the colorigenic assay as it did not respond to unspiked human plasma samples.