Outbreaks of foodborne diseases have become a global health concern; hence, many improvements and developments have been made to reduce the risk of food contamination. We developed a centrifugal microfluidic automatic wireless endpoint detection system integrated with loop mediated isothermal amplification (LAMP) for monoplex pathogen detection. Six identical sets were designed on the microfluidic compact disc (CD) to perform 30 genetic analyses of three different species of foodborne pathogens. The consecutive loading, mixing, and aliquoting of the LAMP primers/reagents and DNA sample solutions were accomplished using an optimized square-wave microchannel, metering chambers and revulsion per minute (RPM) control. We tested 24 strains of pathogenic bacteria (Escherichia coli, Salmonella spp and Vibrio cholerae), with 8 strains of each bacterium, and performed DNA amplification on the microfluidic CD for 60min. Then, the amplicons of the LAMP reaction were detected using the calcein colorimetric method and further analysed via the developed electronic system interfaced with Bluetooth wireless technology to transmit the results to a smartphone. The system showed a limit of detection (LOD) of 3 × 10-5ngμL-1 DNA by analysing the colour change when tested with chicken meat spiked with the three pathogenic bacteria. Since the entire process was performed in a fully automated way and was easy to use, our microdevice is suitable for point-of-care (POC) testing with high simplicity, providing affordability and accessibility even to poor, resource-limited settings.
Polyoctopamine (POct), an amine-functionalised non-conducting polymer, as the transducer layer in an electrochemical biosensor, is presented. This polymer offers versatile covalent coupling either through thiol linker conjugation, carboxyl or aldehyde functional groups without the requirement of pre- or post-surface activation. The colorectal cancer biomarker carcinoembryonic antigen (CEA) was selected as the target analyte, whilst an antibody and a synthetic binding protein, an Affimer, were used as distinct bioreceptors to demonstrate the versatility of polyoctopamine as a transducer polymer layer for oriented immobilisation of the bioreceptors. The electrodeposited polymer layer was characterised using cyclic voltammetry, electrochemical impedance spectroscopy, and on-sensor chemiluminescent blotting. The performance of optimised POct-based biosensors were tested in spiked human serum. Results showed that the electropolymerisation of octopamine on screen printed gold electrode generates a thin polymer film with low resistance. Close proximity of the immobilised bioreceptors to the transducer layer greatly enhanced the sensitivity detection. The sensitivity of the smaller monomeric bioreceptor (Affimer, 12.6 kDa) to detect CEA was comparable to the dimeric antibody (150 kDa) with limit of detection at 11.76 fM which is significantly lower than the basal clinical levels of 25 pM. However, the Affimer-based sensor had a narrower dynamic range compared to the immunosensor (1-100 fM vs. 1 fM - 100 nM, respectively). All electrochemical measurements were done in less than 5 min with small sample volumes (10 μl). Hence, polyoctopamine features a simple fabrication of impedimetric biosensors using amine-functionalisation technique, provides rapid response time with enhanced sensitivity and label-free detection.
This study reports on the development of a novel impedimetric immunosensor design using plant-derived antigenic glycoprotein for the detection of dengue virus (DENV) IgG antibodies. The electrochemical immunosensor platform was constructed using screen-printed carbon electrode (SPCE) modified with graphene/titanium dioxide (G/TiO2) nanocomposite to improve the electrode in terms electrochemical performance and specific surface area. A plant-derived dengue envelope domain III (EDIII) protein was used as the antigenic probe protein in this immunosensing strategy. Under optimised sensing conditions, the immunosensor demonstrated high sensitivity towards DENV IgG in a wide linear working range (62.5-2000 ng/mL), with a limit of detection of 2.81 ng/mL. The immunosensor showed high specificity for discriminating DENV IgG against antibodies of other infectious disease, including the closely related Zika virus (ZIKV). The reliability of the immunosensor in serological diagnosis was verified by challenging the immunosensor against serum samples, compared to conventional enzyme-linked immunosorbent assay (ELISA). As shown by its remarkable performance throughout the study, the devised immunosensor is proposed as a reliable and practical diagnostic tool for the serological detection of dengue in realistic applications.
Keratinocyte traction forces play a crucial role in wound healing. The aim of this study was to develop a novel cell traction force (CTF) transducer system based on cholesteryl ester liquid crystals (LC). Keratinocytes cultured on LC induced linear and isolated deformation lines in the LC surface. As suggested by the fluorescence staining, the deformation lines appeared to correlate with the forces generated by the contraction of circumferential actin filaments which were transmitted to the LC surface via the focal adhesions. Due to the linear viscoelastic behavior of the LC, Hooke's equation was used to quantify the CTFs by associating Young's modulus of LC to the cell induced stresses and biaxial strain in forming the LC deformation. Young's modulus of the LC was profiled by using spherical indentation and determined at approximately 87.1±17.2kPa. A new technique involving cytochalasin-B treatment was used to disrupt the intracellular force generating actin fibers, and consequently the biaxial strain in the LC induced by the cells was determined. Due to the improved sensitivity and spatial resolution (∼1μm) of the LC based CTF transducer, a wide range of CTFs was determined (10-120nN). These were found to be linearly proportional to the length of the deformations. The linear relationship of CTF-deformations was then applied in a bespoke CTF mapping software to estimate CTFs and to map CTF fields. The generated CTF map highlighted distinct distributions and different magnitude of CTFs were revealed for polarized and non-polarized keratinocytes.
Early cancer detection and treatment is an emerging and fascinating field of plasmonic nanobiosensor research. It paves to enrich a life without affecting living cells leading to a possible survival of the patient. This review describes a past and future prospect of an integrated research field on nanostructured metamaterials, microwave transmission, surface plasmonic resonance, nanoantennas, and their manifested versatile properties with nano-biosensors towards early cancer detection to preserve human health. Interestingly, (i) microwave transmission shows more advantages than other electromagnetic radiation in reacting with biological tissues, (ii) nanostructured metamaterial (Au) with special properties like size and shape can stimulate plasmonic effects, (iii) plasmonic based nanobiosensors are to explore the efficacy for early cancer tumour detection or single molecular detection and (iv) nanoantenna wireless communication by using microwave inverse scattering nanomesh (MISN) technique instead of conventional techniques can be adopted to characterize the microwave scattered signals from the biomarkers. It reveals that the nanostructured material with plasmonic nanobiosensor paves a fascinating platform towards early detection of cancer tumour and is anticipated to be exploited as a magnificent field in the future.
Cancer is a leading cause of death globally and early diagnosis is of paramount importance for identifying appropriate treatment pathways to improve cancer patient survival. However, conventional methods for cancer detection such as biopsy, CT scan, magnetic resonance imaging, endoscopy, X-ray and ultrasound are limited and not efficient for early cancer detection. Advancements in molecular technology have enabled the identification of various cancer biomarkers for diagnosis and prognosis of the deadly disease. The detection of these biomarkers can be done by biosensors. Biosensors are less time consuming compared to conventional methods and has the potential to detect cancer at an earlier stage. Compared to conventional biosensors, photoelectrochemical (PEC) biosensors have improved selectivity and sensitivity and is a suitable tool for detecting cancer agents. Recently, 2D carbon materials have gained interest as a PEC sensing platform due to their high surface area and ease of surface modifications for improved electrical transfer and attachment of biorecognition elements. This review will focus on the development of 2D carbon nanomaterials as electrode platform in PEC biosensors for the detection of cancer biomarkers. The working principles, biorecognition strategies and key parameters that influence the performance of the biosensors will be critically discussed. In addition, the potential application of PEC biosensor in clinical settings will also be explored, providing insights into the future perspective and challenges of exploiting PEC biosensors for cancer diagnosis.
Graphene is a 2-dimensional nanomaterial with an atomic thickness has attracted a strong scientific interest owing to their remarkable optical, electronic, thermal, mechanical and electrochemical properties. Graphene-based materials particularly graphene oxide and reduced graphene oxide are widely utilized in various applications ranging from food industry, environmental monitoring and biomedical fields as well as in the development of various types of biosensing devices. The richness in oxygen functional groups in the materials serves as a catalysis for the development of biosensors/electrochemical biosensors which promotes for an attachment of biological recognition elements, surface functionalization and compatible with micro- and nano- bio-environment. In this review, the graphene-based materials application in electrochemical biosensors based on recent advancement (e.g; the surface modification and analytical performances) and the utilization of such biosensors to monitor the noncommunicable diseases are presented. The detection performances of the graphene-based electrochemical biosensors are in the range of ng/mL and have reached up to fg/mL in detecting the targets of NCDs with higher selectivity, sensitivity and stability with good reproducibility attributes. We have discussed the advances while addressing the very specific biomarkers for the NCDs detection. Challenges and possible future research directions for the NCDs detection based on graphene nanocomposite with other 2D nanomaterials are outlined.
A non-enzymatic glucose sensor of multi-walled carbon nanotube-ruthenium oxide/composite paste electrode (MWCNT-RuO(2)/CPE) was developed. The electrode was characterized by using XRD, SEM, TEM and EIS. Meanwhile, cyclic voltammetry and amperometry were used to check on the performances of the MWCNT-RuO(2)/CPE towards glucose. The proposed electrode has displayed a synergistic effect of RuO(2) and MWCNT on the electrocatalytic oxidation of glucose in 3M NaOH. This was possible via the formation of transitions of two redox pairs, viz. Ru(VI)/Ru(IV) and Ru(VII)/Ru(VI). A linear range of 0.5-50mM glucose and a limit of detection of 33 μM glucose (S/N=3) were observed. There was no significant interference observable from the traditional interferences, viz. ascorbic acid and uric acid. Indeed, results so obtained have indicated that the developed MWCNT-RuO(2)/CPE would pave the way for a better future to glucose sensor development as its fabrication was without the use of any enzyme.
Surface acoustic wave mediated transductions have been widely used in the sensors and actuators applications. In this study, a shear horizontal surface acoustic wave (SHSAW) was used for the detection of food pathogenic Escherichia coli O157:H7 (E.coli O157:H7), a dangerous strain among 225 E. coli unique serotypes. A few cells of this bacterium are able to cause young children to be most vulnerable to serious complications. Presence of higher than 1cfu E.coli O157:H7 in 25g of food has been considered as a dangerous level. The SHSAW biosensor was fabricated on 64° YX LiNbO3 substrate. Its sensitivity was enhanced by depositing 130.5nm thin layer of SiO2 nanostructures with particle size lesser than 70nm. The nanostructures act both as a waveguide as well as a physical surface modification of the sensor prior to biomolecular immobilization. A specific DNA sequence from E. coli O157:H7 having 22 mers as an amine-terminated probe ssDNA was immobilized on the thin film sensing area through chemical functionalization [(CHO-(CH2)3-CHO) and APTES; NH2-(CH2)3-Si(OC2H5)3]. The high-performance of sensor was shown with the specific oligonucleotide target and attained the sensitivity of 0.6439nM/0.1kHz and detection limit was down to 1.8femto-molar (1.8×10(-15)M). Further evidence was provided by specificity analysis using single mismatched and complementary oligonucleotide sequences.
The unique property of gold nanoparticles (Au NP) to induce colour change and the versatility of graphene oxides (GO) in surface modification makes them ideal in the application of colorimetric biosensor. Thus we developed a label free optical method to detect DNA hybridization through a visually observed colour change. The Au NP is conjugated to a DNA probe and is allowed to hybridize with the DNA target to the GO thus causing a change in colour from pinkish-red to purplish blue. Spectrophometry analysis gave a wavelength shift of 22 nm with 1 µM of DNA target. Sensitivity testing using serially diluted DNA conjugated GO showed that the optimum detection was at 63 nM of DNA target with the limit at 8 nM. This proves the possibility for the detection of DNA hybridization through the use of dual nanoparticle system by visual observation.
Nanowire sensors offer great potential as highly sensitive electrochemical and electronic biosensors because of their small size, high aspect ratios, and electronic properties. Nevertheless, the available methods to fabricate carbon nanowires in a controlled manner remain limited to expensive techniques. This paper presents a simple fabrication technique for sub-100 nm suspended carbon nanowire sensors by integrating electrospinning and photolithography techniques. Carbon Microelectromechanical Systems (C-MEMS) fabrication techniques allow fabrication of high aspect ratio carbon structures by patterning photoresist polymers into desired shapes and subsequent carbonization of resultant structures by pyrolysis. In our sensor platform, suspended nanowires were deposited by electrospinning while photolithography was used to fabricate support structures. We have achieved suspended carbon nanowires with sub-100 nm diameters in this study. The sensor platform was then integrated with a microfluidic chip to form a lab-on-chip device for label-free chemiresistive biosensing. We have investigated this nanoelectronics label-free biosensor's performance towards bacterial sensing by functionalization with Salmonella-specific aptamer probes. The device was tested with varying concentrations of Salmonella Typhimurium to evaluate sensitivity and various other bacteria to investigate specificity. The results showed that the sensor is highly specific and sensitive in detection of Salmonella with a detection limit of 10 CFU mL-1. Moreover, this proposed chemiresistive assay has a reduced turnaround time of 5 min and sample volume requirement of 5 µL which are much less than reported in the literature.
The application of antibodies in enzyme-linked immunosorbent assay (ELISA) is the basis of this diagnostic technique which is designed to detect a potpourri of complex target molecules such as cell surface antigens, allergens, and food contaminants. However, development of the systematic evolution of Ligands by Exponential Enrichment (SELEX) method, which can generate a nucleic acid-based probe (aptamer) that possess numerous advantages compared to antibodies, offers the possibility of using aptamers as an alternative molecular recognition element in ELISA. Compared to antibodies, aptamers are smaller in size, can be easily modified, are cheaper to produce, and can be generated against a wide array of target molecules. The application of aptamers in ELISA gives rise to an ELISA-derived assay called enzyme-linked apta-sorbent assay (ELASA). As with the ELISA method, ELASA can be used in several different configurations, including direct, indirect, and sandwich assays. This review provides an overview of the strategies involved in aptamer-based ELASA.
The ability of a diagnostic test to detect multiple pathogens simultaneously is useful to obtain meaningful information for clinical treatment and preventive measures. We report a highly sensitive and specific electrochemical biosensor assay for simultaneous detection of three gene targets using quantum dots (QDs). The targets are novel non-protein coding RNA (npcRNA) sequences of Vibrio cholerae, Salmonella sp. and Shigella sp., which cause diarrheal diseases. QDs (PbS, CdS, ZnS) were synthesized and functionalized with DNA probes that were specific to each pathogen. Electrochemical detection of QDs was performed using square wave anodic stripping voltammetry (SWASV). The QDs gave distinct peaks at 0.5 V (PbS), 0.75 V (CdS) and 1.1 V (ZnS). There was no interference in signal response when all three QDs were mixed and detected simultaneously. The detection limits of single and multiplex assays with linear targets and PCR products were in the attomolar ranges. The high assay sensitivity, in combination with specific npcRNA sequences as novel diagnostic targets, makes it a viable tool for detecting pathogens from food, environment and clinical samples.
The non-structural 1 (NS1) protein of the dengue virus circulates in infected patients' blood samples and can be used for early diagnosis of dengue infection. In this paper, we present the detection of naturally-occurring dengue NS1 antigen in infected patient blood plasma using straight long-range surface plasmon waveguides. Three commercially-available anti-NS1 monoclonal antibodies were used for recognition and their performance was compared and discussed. A similar figure of merit to the one used in conventional dengue NS1 capture using an enzyme-linked immunosorbent assay (ELISA) was applied to our results. In general, the positive patient samples can be clearly differentiated from the negative ones and the results agree with those obtained using ELISA. The largest signal-to-noise ratio observed during the experiments was 356 and the best detection limit observed is estimated as 5.73 pg/mm(2).
In this study, we developed a nucleic acid-sensing platform in which a simple, dry-reagent-based nucleic acid amplification assay is combined with a portable multiplex electrochemical genosensor. Preparation of an amplification reaction mix targeting multiple DNA regions of interest is greatly simplified because the lyophilized reagents need only be reconstituted with ultrapure water before the DNA sample is added. The presence of single or multiple target DNAs causes the corresponding single-stranded DNA (ssDNA) amplicons to be generated and tagged with a fluorescein label. The fluorescein-labeled ssDNA amplicons are then analyzed using capture probe-modified screen-printed gold electrode bisensors. Enzymatic amplification of the hybridization event is achieved through the catalytic production of electroactive α-naphthol by anti-fluorescein-conjugated alkaline phosphatase. The applicability of this platform as a diagnostic tool is demonstrated with the detection of toxigenic Vibrio cholerae serogroups O1 and O139, which are associated with cholera epidemics and pandemics. The platform showed excellent diagnostic sensitivity and specificity (100%) when challenged with 168 spiked stool samples. The limit of detection was low (10 colony-forming units/ml) for both toxigenic V. cholerae serogroups. A heat stability assay revealed that the dry-reagent amplification reaction mix was stable at temperatures of 4-56 °C, with an estimated shelf life of seven months. The findings of this study highlight the potential of combining a dry-reagent-based nucleic acid amplification assay with an electrochemical genosensor in a more convenient, sensitive, and sequence-specific detection strategy for multiple target nucleic acids.
D-serine has been implicated as a brain messenger, promoting not only neuronal signalling but also synaptic plasticity. Thus, a sensitive tool for D-serine monitoring in brain is required to understand the mechanisms of D-serine release from glia cells. A biosensor for direct fixed potential amperometric monitoring of D-serine incorporating mammalian D-amino acid oxidase (DAAO) immobilized on a Nafion coated poly-ortho-phenylenediamine (PPD) modified Pt-Ir disk electrode was therefore developed. The combined layers of PPD and Nafion enhanced the enzyme activity and biosensor efficiency by approximately 2-fold compared with each individual layer. A steady state response time (t(90%)) of 0.7+/-0.1s (n=8) and limit of detection 20+/-1 nM (n=8) were obtained. Cylindrical geometry showed lower sensitivity compared to disk geometry (61+/-7 microA cm(-2) mM(-1), (n=4), R(2)=0.999). Interference by ascorbic acid (AA), the main interference species in the central nervous system and other neurochemical electroactive molecules was negligible. Implantation of the electrode and microinjection of D-serine into rat brain striatal extracellular fluid demonstrated that the electrode was capable of detecting D-serine in brain tissue in vivo.