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.
A novel method for the rapid modification of fullerene for subsequent enzyme attachment to create a potentiometric biosensor is presented. Urease was immobilized onto the modified fullerene nanomaterial. The modified fullerene-immobilized urease (C60-urease) bioconjugate has been confirmed to catalyze the hydrolysis of urea in solution. The biomaterial was then deposited on a screen-printed electrode containing a non-plasticized poly(n-butyl acrylate) (PnBA) membrane entrapped with a hydrogen ionophore. This pH-selective membrane is intended to function as a potentiometric urea biosensor with the deposition of C60-urease on the PnBA membrane. Various parameters for fullerene modification and urease immobilization were investigated. The optimal pH and concentration of the phosphate buffer for the urea biosensor were 7.0 and 0.5 mM, respectively. The linear response range of the biosensor was from 2.31 × 10-3 M to 8.28 × 10-5 M. The biosensor's sensitivity was 59.67 ± 0.91 mV/decade, which is close to the theoretical value. Common cations such as Na+, K+, Ca2+, Mg2+ and NH4+ showed no obvious interference with the urea biosensor's response. The use of a fullerene-urease bio-conjugate and an acrylic membrane with good adhesion prevented the leaching of urease enzyme and thus increased the stability of the urea biosensor for up to 140 days.
In the bacteria kingdom, quorum sensing (QS) is a cell-to-cell communication that relies on the production of and response to specific signaling molecules. In proteobacteria, N-acylhomoserine lactones (AHLs) are the well-studied signaling molecules. The present study aimed to characterize the production of AHL of a bacterial strain A9 isolated from a Malaysian tropical soil. Strain A9 was identified as Burkholderia sp. using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry and 16S rDNA nucleotide sequence analysis. AHL production by A9 was detected with two biosensors, namely Chromobacterium violaceum CV026 and Escherichia coli [pSB401]. Thin layer chromatography results showed N-hexanoylhomoserine lactone (C6-HSL) and N-octanoylhomoserine lactone (C8-HSL) production. Unequivocal identification of C6-HSL and C8-HSL was achieved by high resolution triple quadrupole liquid chromatography-mass spectrometry analysis. We have demonstrated that Burkholderia sp. strain A9 produces AHLs that are known to be produced by other Burkholderia spp. with CepI/CepR homologs.
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.
A disposable horseradish peroxidase (HRP)-based electrochemical genosensor was developed for chronoamperometric detection of single-stranded asymmetric lolB gene PCR amplicon (118 bp in length) of the food-borne pathogen, Vibrio cholerae. A two-step sandwich-type hybridization strategy using two specific probes was employed for specific detection of the target single-stranded DNA (ssDNA). The analytical performances of the detection platform have been evaluated using a synthetic ssDNA (ST3) which was identical to the target single-stranded amplicon and a total of 19 bacterial strains. Under optimal condition, ST3 was calibrated with a dynamic range of 0.4883-15.6250 nM. By coupling asymmetric PCR amplification, the probe-based electrochemical genosensor was highly specific to the target organism (100% specificity) and able to detect as little as 0.85 ng/μl of V. cholerae genomic DNA.
Fabrication of a test strip for detection of benzoic acid was successfully implemented by immobilizing tyrosinase, phenol and 3-methyl-2-benzothiazolinone hydrazone (MBTH) onto filter paper using polystyrene as polymeric support. The sensing scheme was based on the decreasing intensity of the maroon colour of the test strip when introduced into benzoic acid solution. The test strip was characterized using optical fiber reflectance and has maximum reflectance at 375 nm. It has shown a highly reproducible measurement of benzoic acid with a calculated RSD of 0.47% (n = 10). The detection was optimized at pH 7. A linear response of the biosensor was obtained in 100 to 700 ppm of benzoic acid with a detection limit (LOD) of 73.6 ppm. At 1:1 ratio of benzoic acid to interfering substances, the main interfering substance is boric acid. The kinetic analyses show that, the inhibition of benzoic is competitive inhibitor and the inhibition constant (K(i)) is 52.9 ppm. The activity of immobilized tyrosinase, phenol, and MBTH in the test strip was fairly sustained during 20 days when stored at 3 °C. The developed test strip was used for detection of benzoic acid in food samples and was observed to have comparable results to the HPLC method, hence the developed test strip can be used as an alternative to HPLC in detecting benzoic acid in food products.
Behavioural assessment of experimental pain is an essential method for analysing and measuring pain levels. Rodent models, which are widely used in behavioural tests, are often subject to external forces and stressful manipulations that cause variability of the parameters measured during the experiment. Therefore, these parameters may be inappropriate as indicators of pain. In this article, a stepping-force analgesimeter was designed to investigate the variations in the stepping force of rats in response to pain induction. The proposed apparatus incorporates new features, namely an infrared charge-coupled device (CCD) camera and a data acquisition system. The camera was able to capture the locomotion of the rats and synchronise the stepping force concurrently so that each step could be identified. Inter-day and intra-day precision and accuracy of each channel (there were a total of eight channels in the analgesimeter and each channel was connected to one load cell and one amplifier) were studied using different standard load weights. The validation studies for each channel also showed convincing results whereby intra-day and inter-day precision were less than 1% and accuracy was 99.36-100.36%. Consequently, an in vivo test was carried out using 16 rats (eight females and eight males). The rats were allowed to randomly walk across the sensor tunnel (the area that contained eight channels) and the stepping force and locomotion were recorded. A non-expert, but from a related research domain, was asked to differentiate the peaks of the front and hind paw, respectively. The results showed that of the total movement generated by the rats, 50.27 ± 3.90% in the case of the male rats and 62.20 ± 6.12% in that of the female rats had more than two peaks, a finding which does not substantiate the assumptions made in previous studies. This study also showed that there was a need to use the video display frame to distinguish between the front and hind paws in the case of 48.80 ± 4.01% of the male rats and 66.76 ± 5.35% of the female rats. Evidently the assumption held by current researchers regarding stepping force measurement is not realistic in terms of application, and as this study has shown, the use of a video display frame is essential for the identification of the front and hind paws through the peak signals.
A new implantable electrocatalytic glucose sensor for subcutaneous glucose monitoring has been fabricated by immobilizing glucose oxidase on a chemically modified carbon fiber. The sensor was inserted subcutaneously on a male spraguely rat without any incision after dipping the microsensor in the rat's serum for 3 days. The so called "stained" microsensor, operated in the amperometric mode with an applied potential of +0.23 V versus Ag|AgCl, was able to directly measure the glucose concentration upon infusion of glucose. The results obtained were encouraging, with the response time was less than 2s and the apparent Michaelis-Menten value at 5.1+/-0.5mM. The "stained" microsensor shows good stability and reproducibility with constant response spanned over 25 days. Most common interferences in glucose analysis were minimized by the outerlayer Nafion. Hematology examinations showed minimal material-tissue interaction. Use of such mechanical devices will allow a more refined understanding towards glucose control in diabetic patients as the implanted microsensor was not effected by biocompatibility failures.
This paper presents a comparison between data from single modality and fusion methods to classify Tualang honey as pure or adulterated using Linear Discriminant Analysis (LDA) and Principal Component Analysis (PCA) statistical classification approaches. Ten different brands of certified pure Tualang honey were obtained throughout peninsular Malaysia and Sumatera, Indonesia. Various concentrations of two types of sugar solution (beet and cane sugar) were used in this investigation to create honey samples of 20%, 40%, 60% and 80% adulteration concentrations. Honey data extracted from an electronic nose (e-nose) and Fourier Transform Infrared Spectroscopy (FTIR) were gathered, analyzed and compared based on fusion methods. Visual observation of classification plots revealed that the PCA approach able to distinct pure and adulterated honey samples better than the LDA technique. Overall, the validated classification results based on FTIR data (88.0%) gave higher classification accuracy than e-nose data (76.5%) using the LDA technique. Honey classification based on normalized low-level and intermediate-level FTIR and e-nose fusion data scored classification accuracies of 92.2% and 88.7%, respectively using the Stepwise LDA method. The results suggested that pure and adulterated honey samples were better classified using FTIR and e-nose fusion data than single modality data.
Human chorionic gonadotropin (hCG), a glycoprotein hormone secreted from the placenta, is a key molecule that indicates pregnancy. Here, we have designed a cost-effective, label-free, in situ point-of-care (POC) immunosensor to estimate hCG using a cuneated 25 nm polysilicon nanogap electrode. A tiny chip with the dimensions of 20.5 × 12.5 mm was fabricated using conventional lithography and size expansion techniques. Furthermore, the sensing surface was functionalized by (3-aminopropyl)triethoxysilane and quantitatively measured the variations in hCG levels from clinically obtained human urine samples. The dielectric properties of the present sensor are shown with a capacitance above 40 nF for samples from pregnant women; it was lower with samples from non-pregnant women. Furthermore, it has been proven that our sensor has a wide linear range of detection, as a sensitivity of 835.88 μA mIU(-1) ml(-2) cm(-2) was attained, and the detection limit was 0.28 mIU/ml (27.78 pg/ml). The dissociation constant Kd of the specific antigen binding to the anti-hCG was calculated as 2.23 ± 0.66 mIU, and the maximum number of binding sites per antigen was Bmax = 22.54 ± 1.46 mIU. The sensing system shown here, with a narrow nanogap, is suitable for high-throughput POC diagnosis, and a single injection can obtain triplicate data or parallel analyses of different targets.
The discovery of Systematic Evolution of Ligands by Exponential Enrichment (SELEX) assay has led to the generation of aptamers from libraries of nucleic acids. Concomitantly, aptamer-target recognition and its potential biomedical applications have become a major research endeavour. Aptamers possess unique properties that make them superior biological receptors to antibodies with a plethora of target molecules. Some specific areas of opportunities explored for aptamer-target interactions include biochemical analysis, cell signalling and targeting, biomolecular purification processes, pathogen detection and, clinical diagnosis and therapy. Most of these potential applications rely on the effective immobilisation of aptamers on support systems to probe target species. Hence, recent research focus is geared towards immobilising aptamers as oligosorbents for biodetection and bioscreening. This article seeks to review advances in immobilised aptameric binding with associated successful milestones and respective limitations. A proposal for high throughput bioscreening using continuous polymeric adsorbents is also presented.
A top-down nanofabrication approach is used to develop silicon nanowires from silicon-on-insulator (SOI) wafers and involves direct-write electron beam lithography (EBL), inductively coupled plasma-reactive ion etching (ICP-RIE) and a size reduction process. To achieve nanometer scale size, the crucial factors contributing to the EBL and size reduction processes are highlighted. The resulting silicon nanowires, which are 20 nm in width and 30 nm in height (with a triangular shape) and have a straight structure over the length of 400 μm, are fabricated precisely at the designed location on the device. The device is applied in biomolecule detection based on the changes in drain current (Ids), electrical resistance and conductance of the silicon nanowires upon hybridization to complementary target deoxyribonucleic acid (DNA). In this context, the scaled-down device exhibited superior performances in terms of good specificity and high sensitivity, with a limit of detection (LOD) of 10 fM, enables for efficient label-free, direct and higher-accuracy DNA molecules detection. Thus, this silicon nanowire can be used as an improved transducer and serves as novel biosensor for future biomedical diagnostic applications.
We have developed a colorimetric biosensor using a dual platform of gold nanoparticles and graphene oxide sheets for the detection of Salmonella enterica. The presence of the invA gene in S. enterica causes a change in color of the biosensor from its original pinkish-red to a light purplish solution. This occurs through the aggregation of the primary gold nanoparticles-conjugated DNA probe onto the surface of the secondary graphene oxide-conjugated DNA probe through DNA hybridization with the targeted DNA sequence. Spectrophotometry analysis showed a shift in wavelength from 525 nm to 600 nm with 1 μM of DNA target. Specificity testing revealed that the biosensor was able to detect various serovars of the S. enterica while no color change was observed with the other bacterial species. Sensitivity testing revealed the limit of detection was at 1 nM of DNA target. This proves the effectiveness of the biosensor in the detection of S. enterica through DNA hybridization.
An amperometric enzyme-electrode was introduced where glucose oxidase (GOD) was immobilized on chitosan membrane via crosslinking, and then fastened on a platinum working electrode. The immobilized enzyme showed relatively high retention activity. The activity of the immobilized enzyme was influenced by its loading, being suppressed when more than 0.6 mg enzyme was used in the immobilization. The biosensor showing the highest response to glucose utilized 0.21 ml/cm2 thick chitosan membrane. The optimum experimental conditions for the biosensors in analysing glucose dissolved in 0.1 M phosphate buffer (pH 6.0) were found to be 35°C and 0.6 V applied potential. The introduced biosensor reached a steady-state current at 60 s. The apparent Michaelis-Menten constant ([Formula: see text]) of the biosensor was 14.2350 mM, and its detection limit was 0.05 mM at s/n > 3, determined experimentally. The RSD of repeatability and reproducibility of the biosensor were 2.30% and 3.70%, respectively. The biosensor was showed good stability; it retained ~36% of initial activity after two months of investigation. The performance of the biosensors was evaluated by determining the glucose content in fruit homogenates. Their accuracy was compared to that of a commercial glucose assay kit. There was no significance different between two methods, indicating the introduced biosensor is reliable.
The pragmatic outcome of a lung cancer diagnosis is closely interrelated in reducing the number of fatal death caused by the world's top cancerous disease. Regardless of the advancement made in understanding lung tumor, and its multimodal treatment, in general the percentage of survival remain low. Late diagnosis of a cancerous cell in patients is the major hurdle for the above circumstances. In the new era of a lung cancer diagnosis with low cost, portable and non-invasive clinical sampling, nanotechnology is at its inflection point where current researches focus on the implementation of biosensor conjugated nanomaterials for the generation of the ideal sensing. The present review encloses the superiority of nanomaterials from zero to three-dimensional nanostructures in its discrete and nanocomposites nanotopography on sensing lung cancer biomarkers. Recent researches conducted on definitive nanomaterials and nanocomposites at multiple dimension with distinctive physiochemical property were focused to subside the cases associated with lung cancer through the development of novel biosensors. The hurdles encountered in the recent research and future preference with prognostic clinical lung cancer diagnosis using multidimensional nanomaterials and its composites are presented.
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.
The volatile compounds of pork, other meats and meat products were studied using an electronic nose and gas chromatography mass spectrometer with headspace analyzer (GCMS-HS) for halal verification. The zNose™ was successfully employed for identification and differentiation of pork and pork sausages from beef, mutton and chicken meats and sausages which were achieved using a visual odor pattern called VaporPrint™, derived from the frequency of the surface acoustic wave (SAW) detector of the electronic nose. GCMS-HS was employed to separate and analyze the headspace gasses from samples into peaks corresponding to individual compounds for the purpose of identification. Principal component analysis (PCA) was applied for data interpretation. Analysis by PCA was able to cluster and discriminate pork from other types of meats and sausages. It was shown that PCA could provide a good separation of the samples with 67% of the total variance accounted by PC1.
Graphene decorated with graphitic nanospheres functionalized with pyrene butyric acid (PBA) is used for the first time to fabricate a DNA biosensor. The electrode was formed by attaching a DNA probe onto PBA, which had been stacked onto a graphene material decorated with graphene nanospheres (GNSs). The nanomaterial was drop-coated onto a carbon screen-printed electrode (SPE) to create the GNS-PBA modified electrode (GNS-PBA/SPE). A simple method was used to produce GNS by annealing graphene oxide (GO) solution at high temperature. Field emission scanning electron micrographs confirmed the presence of a spherical shape of GNS with a diameter range of 40-80 nm. A stable and uniform PBA-modified GNS (GNS-PBA) was obtained with a facile ultrasonication step. Thus allowing aminated DNA probes of genetically modified (GM) soybean to be attached to the nanomaterials to form the DNA biosensor. The GNS-PBA/SPE exhibited excellent electrical conductivity via cyclic voltammetry (CV) and differential pulse voltammetry (DPV) tests using potassium ferricyanide (K3[Fe(CN)6]) as the electroactive probe. By employing an anthraquinone monosulfonic acid (AQMS) redox intercalator as the DNA hybridization indicator, the biosensor response was evaluated using the DPV electrochemical method. A good linear relationship between AQMS oxidation peak current and target DNA concentrations from 1.0 × 10-16 to 1.0 × 10-8 M with a limit of detection (LOD) of less than 1.0 × 10-16 M was obtained. Selectivity experiments revealed that the voltammetric GM DNA biosensor could discriminate complementary sequences of GM soybean from non-complementary sequences and hence good recoveries were obtained for real GM soybean sample analysis. The main advantage of using GNS is an improvement of the DNA biosensor analytical performance.
Cardiovascular disease is a major global health issue. In particular, acute myocardial infarction (AMI) requires urgent attention and early diagnosis. The use of point-of-care diagnostics has resulted in the improved management of cardiovascular disease, but a major drawback is that the performance of POC devices does not rival that of central laboratory tests. Recently, many studies and advances have been made in the field of surface-enhanced Raman scattering (SERS), including the development of POC biosensors that utilize this detection method. Here, we present a review of the strengths and limitations of these emerging SERS-based biosensors for AMI diagnosis. The ability of SERS to multiplex sensing against existing POC detection methods are compared and discussed. Furthermore, SERS calibration-free methods that have recently been explored to minimize the inconvenience and eliminate the limitations caused by the limited linear range and interassay differences found in the calibration curves are outlined. In addition, the incorporation of artificial intelligence (AI) in SERS techniques to promote multivariate analysis and enhance diagnostic accuracy are discussed. The future prospects for SERS-based POC devices that include wearable POC SERS devices toward predictive, personalized medicine following the Fourth Industrial Revolution are proposed.