Lateral flow assays (LFAs) have currently attracted broad interest for point-of-care (POC) diagnostics, but their application has been restricted by poor quantification and limited sensitivity. While the former has been currently solved to some extent by the development of handheld or smartphone-based readers, the latter has not been addressed fully, particularly the potential influences of environmental conditions (e.g., temperature and relative humidity (RH)), which have not yet received serious attention. The present study reports the use of a portable temperature-humidity control device to provide an optimum environmental requirement for sensitivity improvement in LFAs, followed by quantification by using a smartphone. We found that a RH beyond 60% with temperatures of 55-60°C and 37-40°C produced optimum nucleic acid hybridization and antigen-antibody interaction in LFAs, respectively representing a 10-fold and 3-fold signal enhancement over ambient conditions (25°C, 60% RH). We envision that in the future the portable device could be coupled with a fully integrated paper-based sample-to-answer biosensor for sensitive detection of various target analytes in POC settings.
Human immunodeficiency virus (HIV) has infected almost 35 million people worldwide. Various tests have been developed to detect the presence of HIV during the early stages of the disease in order to reduce the risk of transmission to other humans. The HIV-1 Tat protein is one of the proteins present in HIV that are released abundantly approximately 2-4 weeks after infection. In this review, we have outlined various strategies for detecting the Tat protein, which helps transcribe the virus and enhances replication. Detection strategies presented include immunoassays, biosensors and gene expression, which utilize antibodies or aptamers as common probes to sense the presence of Tat. Alternatively, measuring the levels of gene transcription is a direct method of analysing the HIV gene to confirm the presence of Tat. By detection of the Tat protein, virus transmission can be detected in high-risk individuals in the early stages of the disease to reduce the risk of an HIV pandemic.
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, a disposable and simple electrochemical immunosensor was fabricated for the detection of carcinoembryonic antigen. In this method, silver nanoparticles (AgNPs) were mixed with reduced graphene oxide (rGO) to modify the surface of screen-printed carbon electrode (SPE). Initially, AgNPs-rGO modified-SPEs were fabricated by using simple electrochemical deposition method. Then the carcinoembryonic antigen (CEA) was immobilized between the primary antibody and horseradish peroxidase (HRP)-conjugated secondary antibody onto AgNPs-rGO modified-SPEs to fabricate a sandwich-type electrochemical immunosensor. The proposed method could detect the CEA with a linear range of 0.05-0.50µgmL-1 and a detection limit down to 0.035µgmL-1 as compared to its non-sandwich counterpart, which yielded a linear range of 0.05-0.40µgmL-1, with a detection limit of 0.042µgmL-1. The immunosensor showed good performance in the detection of carcinoembryonic antigen, exhibiting a simple, rapid and low-cost. The immunosensor showed a higher sensitivity than an enzymeless sensor.
A quartz crystal microbalance (QCM) sensor platform was used to develop an immunosensor for the detection of food pathogen Campylobacter jejuni. Rabbit polyclonal antibodies and commercially available mouse monoclonal antibodies against C. jejuni were investigated to construct direct, sandwich and gold-nanoparticles (AuNPs) amplified sandwich assays. The performance of the QCM immunosensor developed using sandwich assay by utilising the rabbit polyclonal antibody as the capture antibody and conjugated to AuNPs as the detection antibody gave the highest sensitivity. This sensor achieved a limit of detection (LOD) of 150 colony forming unit (CFU)mL(-1) of C. jejuni in solution. The QCM sensor showed excellent sensitivity and specificity for Campylobacter detection with low cross reactivity for other foodborne pathogens such as Salmonella Typhimurium, (7%) Listeria monocytogenes (3%) and Escherichia coli (0%). The development of this biosensor would help in the sensitive detection of Campylobacter which can result in reducing pre-enrichment steps; hence, reducing assay time. This work demonstrates the potential of this technology for the development of a rapid and sensitive detection method for C. jejuni.
We describe a gold nanoparticle-based sandwich immunoassay for the dual detection and measurement of hemoglobin A1c (HbA1c) and total hemoglobin in the whole blood (without pretreatment) in a single step for personalized medicine. The optimized antibody-functionalized gold nanoparticles immunoreact simultaneously with HbA1c and total hemoglobin to form a sandwich at distinctive test lines to transduce visible signals. The applicability of this method as a personal management tool was demonstrated by establishing a calibration curve to relate % HbA1c, a useful value for type 2 diabetes management, to the signal ratio of captured HbA1c to all other forms of hemoglobin. The platform showed excellent selectivity (100%) toward HbA1c at distinctive test lines when challenged with HbA0, glycated HbA0 and HbA2. The reproducibility of the measurement was good (6.02%) owing to the dual measurement of HbA1c and total hemoglobin. A blood sample stability test revealed that the quantitative measurement of % HbA1c was consistent and no false-positive results were detected. Also, this method distinguished the blood sample with elevated HbF from the normal samples and the variants. The findings of this study highlight the potential of a lateral flow immunosensor as a simple, inexpensive, consistent, and convenient strategy for the dual measurement of HbA1c and total Hb to provide useful % HbA1c values for better on-site diabetes care.
Electrospun polyhydroxybutyrate (PHB) fibers were dip-coated by polymethyl methacrylate-co-methacrylic acid, poly(MMA-co-MAA), which was synthesized in different molar ratios of the monomers via free-radical polymerization. Fabricated platfrom was employed for immobilization of the dengue antibody and subsequent detection of dengue enveloped virus in enzyme-linked immunosorbent assay (ELISA). There is a major advantage for combination of electrospun fibers and copolymers. Fiber structre of electrospun PHB provides large specific surface area available for biomolecular interaction. In addition, polymer coated parts of the platform inherited the premanent presence of surface carboxyl (-COOH) groups from MAA segments of the copolymer which can be effectively used for covalent and physical protein immobilization. By tuning the concentration of MAA monomers in polymerization reaction the concentration of surface -COOH groups can be carefully controlled. Therefore two different techniques have been used for immobilization of the dengue antibody aimed for dengue detection: physical attachment of dengue antibodies to the surface and covalent immobilization of antibodies through carbodiimide chemistry. In that perspective, several different characterization techniques were employed to investigate the new polymeric fiber platform such as scanning electron microscopy (SEM), atomic force microscopy (AFM), water contact angle (WCA) measurement and UV-vis titration. Regardless of the immobilization techniques, substantially higher signal intensity was recorded from developed platform in comparison to the conventional ELISA assay.
The study demonstrates the development of a liquid-based gate-control silicon nanowire biosensor for detection of specific single-stranded DNA (ssDNA) molecules. The sensor was fabricated using conventional photolithography coupled with an inductively coupled plasma dry etching process. Prior to the application of DNA to the device, its linear response to pH was confirmed by serial dilution from pH 2 to pH 14. Then, the sensor surface was silanized and directly aminated with (3-aminopropyl) triethoxysilane to create a molecular binding chemistry for biofunctionalization. The resulting Si‒O‒Si‒ components were functionalized with receptor ssDNA, which interacted with the targeted ssDNA to create a field across the silicon nanowire and increase the current. The sensor shows selectivity for the target ssDNA in a linear range from target ssDNA concentrations of 100 pM to 25 nM. With its excellent detection capabilities, this sensor platform is promising for detection of specific biomarkers and other targeted proteins.
Dengue is the current leading cause of death among children in several Latin American and Asian countries. Due to poverty in areas where the disease is prevalent and the high cost of conventional diagnostic systems, low cost devices are needed to reduce the burden caused by dengue infection. Centrifugal microfluidic platforms are an alternative solution to reduce costs and increase the availability of a rapid diagnostic system. The rate of chemical reactions in such devices often depends on the efficiency of the mixing techniques employed in their microfluidic networks. This paper introduces a micromixer that operates by the expansion and contraction of a microballoon to produce a consistent periodical 3D reciprocating flow. We established that microballoons reduced mixing time of 12 μl liquids from 170 min, for diffusional mixing, to less than 23 s. We have also tested the effect of the microballoon mixers on the detection of the dengue virus. The results indicate that employing a microballoon mixer enhances the detection sensitivity of the dengue virus by nearly one order of magnitude compared to the conventional ELISA method.
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.
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.
A novel optical detection system consisting of combination of uricase/HRP-CdS quantum dots (QDs) for the determination of uric acid in urine sample is described. The QDs was used as an indicator to reveal fluorescence property of the system resulting from enzymatic reaction of uricase and HRP (horseradish peroxidase), which is involved in oxidizing uric acid to allaintoin and hydrogen peroxide. The hydrogen peroxide produced was able to quench the QDs fluorescence, which was proportional to uric acid concentration. The system demonstrated sufficient activity of uricase and HRP at a ratio of 5U:5U and pH 7.0. The linearity of the system toward uric acid was in the concentration range of 125-1000 µM with detection limit of 125 µM.
The illegal administration of recombinant human erythropoietin (rHuEPO) among athletes is largely preferred over blood doping to enhance stamina. The advent of recombinant DNA technology allowed the expression of EPO-encoding genes in several eukaryotic hosts to produce rHuEPO, and today these performance-enhancing drugs are readily available. As a mimetic of endogenous EPO (eEPO), rHuEPO augments the oxygen carrying capacity of blood. Thus, monitoring the illicit use of rHuEPO among athletes is crucial in ensuring an even playing field and maintaining the welfare of athletes. A number of rHuEPO detection methods currently exist, including measurement of hematologic parameters, gene-based detection methods, glycomics, use of peptide markers, electrophoresis, isoelectric focusing (IEF)-double immunoblotting, aptamer/antibody-based methods, and lateral flow tests. This review gleans these different strategies and highlights the leading molecular recognition elements that have potential roles in rHuEPO doping detection.
Influenza viruses, which are RNA viruses belonging to the family Orthomyxoviridae, cause respiratory diseases in birds and mammals. With seasonal epidemics, influenza spreads all over the world, resulting in pandemics that cause millions of deaths. Emergence of various types and subtypes of influenza, such as H1N1 and H7N9, requires effective surveillance to prevent their spread and to develop appropriate anti-influenza vaccines. Diagnostic probes such as glycans, aptamers, and antibodies now allow discrimination among the influenza strains, including new subtypes. Several sensors have been developed based on these probes, efforts made to augment influenza detection. Herein, we review the currently available sensing strategies to detect influenza viruses.
The ubiquitous nature of bacteria enables them to survive in a wide variety of environments. Hence, the rise of various pathogenic species that are harmful to human health raises the need for the development of accurate sensing systems. Sensing systems are necessary for diagnosis and epidemiological control of pathogenic organism, especially in the food-borne pathogen and sanitary water treatment facility' bacterial populations. Bacterial sensing for the purpose of diagnosis can function in three ways: bacterial morphological visualization, specific detection of bacterial component and whole cell detection. This paper provides an overview of the currently available bacterial detection systems that ranges from microscopic observation to state-of-the-art smartphone-based detection.
Sensing applications can be used to report biomolecular interactions in order to elucidate the functions of molecules. The use of an analyte and a ligand is a common set-up in sensor development. For several decades, antibodies have been considered to be potential analytes or ligands for development of so-called "immunosensors." In an immunosensor, formation of the complex between antibody and antigen transduces the signal, which is measurable in various ways (e.g., both labeled and label-free based detection). Success of an immunosensor depends on various factors, including surface functionalization, antibody orientation, density of the antibody on the sensor platform, and configuration of the immunosensor. Careful optimization of these factors can generate clear-cut results for any immunosensor. Herein, current aspects, involved in the generated immunosensors, are discussed.
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.
Advanced diagnostic technologies, such as polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA), have been widely used in well-equipped laboratories. However, they are not affordable or accessible in resource-limited settings due to the lack of basic infrastructure and/or trained operators. Paper-based diagnostic technologies are affordable, user-friendly, rapid, robust, and scalable for manufacturing, thus holding great potential to deliver point-of-care (POC) diagnostics to resource-limited settings. In this review, we present the working principles and reaction mechanism of paper-based diagnostics, including dipstick assays, lateral flow assays (LFAs), and microfluidic paper-based analytical devices (μPADs), as well as the selection of substrates and fabrication methods. Further, we report the advances in improving detection sensitivity, quantification readout, procedure simplification and multi-functionalization of paper-based diagnostics, and discuss the disadvantages of paper-based diagnostics. We envision that miniaturized and integrated paper-based diagnostic devices with the sample-in-answer-out capability will meet the diverse requirements for diagnosis and treatment monitoring at the POC.
In this article we present ultra-sensitive, silicon nanowire (SiNW)-based biosensor devices for the detection of disease biomarkers. An electrochemically induced functionalisation method has been employed to graft antibodies targeted against the prostate cancer risk biomarker 8-hydroxydeoxyguanosine (8-OHdG) to SiNW surfaces. The antibody-functionalised SiNW sensor has been used to detect binding of the 8-OHdG biomarker to the SiNW surface within seconds of exposure. Detection of 8-OHdG concentrations as low as 1 ng/ml (3.5 nM) has been demonstrated. The active device has been bonded to a disposable printed circuit which can be inserted into an electronic readout system as part of an integrated Point of Care (POC) diagnostic. The speed, sensitivity and ease of detection of biomarkers using SiNW sensors render them ideal for eventual POC diagnostics.
Vibrio cholerae is a human pathogen that causes mild to severe diarrheal illnesses and has major public health significance. Herein, we present a thermostabilized electrochemical genosensing assay combining the use of magnetic beads as a biorecognition platform and gold nanoparticles as a hybridization tag for the detection and quantification of V. cholerae lolB gene single-stranded asymmetric PCR amplicons as an alternative to the time-consuming classical isolation method. This thermostabilized, pre-mixed, pre-aliquoted and ready-to-use magnetogenosensing assay simplified the procedures and permitted the reaction to be conducted at room temperature. The asymmetric PCR amplicons were hybridized to a magnetic bead-functionalized capture probe and a fluorescein-labeled detection probe followed by tagging with gold nanoparticles. Electrochemical detection of the chemically dissolved gold nanoparticles was performed using the differential pulse anodic stripping voltammetry method. The real-time stability evaluation of thermostabilized assay was found to be stable for at least 180 days at room temperature (25-30°C). The analytical specificity of the assay was 100%, while its analytical sensitivity was linearly related to different concentrations of 200-mer synthetic target, purified genomic DNA, and bacterial culture with a limit of detection (LoD) of 3.9nM, 5pg/µl, and 10(3)CFU/ml, respectively. The clinical applicability of the assay was successfully validated using spiked stool samples with an average current signal-to-cut-off ratio of 10.8. Overall, the precision of the assay via relative standard deviation was <10%, demonstrating its reliability and accuracy.