Label-free-based detection is pivotal for real-time monitoring of biomolecular interactions and to eliminate the need for labeling with tags that can occupy important binding sites of biomolecules. One simplest form of label-free-based detection is ultraviolet-visible-near-infrared (UV-vis-NIR) spectroscopy, which measure changes in reflectivity as a means to monitor immobilization and interaction of biomolecules with their corresponding partners. In biosensor development, the platform used for the biomolecular interaction should be suitable for different molecular recognition elements. In this study, gold (Au)-coated polycarbonate was used as a platform and as a proof-of-concept, erythropoietin (EPO), a doping substance widely abused by the athletes was used as the target. The interaction of EPO with its corresponding molecular recognition elements (anti-EPO monoclonal antibody and anti-EPO DNA aptamer) is monitored by UV-vis-NIR spectroscopy. Prior to this, to show that UV-vis-NIR spectroscopy is a suitable method for measuring biomolecular interaction, the interaction between biotin and streptavidin was demonstrated via this strategy and reflectivity of this interaction decreased by 25%. Subsequent to this, interaction of the EPO with anti-EPO monoclonal antibody and anti-EPO DNA aptamer resulted in the decrease of reflectivity by 5% and 10%, respectively. The results indicated that Au-coated polycarbonate could be an ideal biosensor platform for monitoring biomolecular interactions using UV-vis-NIR spectroscopy. A smaller version of the Au-coated polycarbonate substrates can be derived from the recent set-up, to be applied towards detecting EPO abuse among atheletes.
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 discovery that synthetic short chain nucleic acids are capable of selective binding to biological targets has made them to be widely used as molecular recognition elements. These nucleic acids, called aptamers, are comprised of two types, DNA and RNA aptamers, where the DNA aptamer is preferred over the latter due to its stability, making it widely used in a number of applications. However, the success of the DNA selection process through Systematic Evolution of Ligands by Exponential Enrichment (SELEX) experiments is very much dependent on its most critical step, which is the conversion of the dsDNA to ssDNA. There is a plethora of methods available in generating ssDNA from the corresponding dsDNA. These include asymmetric PCR, biotin-streptavidin separation, lambda exonuclease digestion and size separation on denaturing-urea PAGE. Herein, different methods of ssDNA generation following the PCR amplification step in SELEX are reviewed.
Aptamers are single stranded DNA or RNA oligonucleotides that have high affinity and specificity towards a wide range of target molecules. Aptamers have low molecular weight, amenable to chemical modifications and exhibit stability undeterred by repetitive denaturation and renaturation. Owing to these indispensable advantages, aptamers have been implemented as molecular recognition element as alternative to antibodies in various assays for diagnostics. By amalgamating with a number of methods that can provide information on the aptamer-target complex formation, aptamers have become the elemental tool for numerous biosensor developments. In this review, administration of aptamers in applications involving assays of fluorescence, electrochemistry, nano-label and nano-constructs are discussed. Although detection strategies are different for various aptamer-based assays, the core of the design strategies is similar towards reporting the presence of specific target binding to the corresponding aptamers. It is prognosticated that aptamers will find even broader applications with the development of new methods of transducing aptamer target binding.
We describe a rare case of vertebra (intraosseous) hemangioma with bilateral and symmetrical epidural extension causing cord compression in a 24-year-old woman. The epidural component was isointense to cord on both T1 and T2 sequences, and enhanced markedly and homogenously following gadolinium administration. The gradual in onset and progressive nature with the typical enhancing pattern lead the neurosurgeon to the more common diagnosis of spinal meningioma. Epidural extension of vertebral hemangiomas causing cord compression is rarely reported. Review of literatures reveal that cases that have been reported are of unilateral extension into epidural space and of cavernous type. This is the first case report of capillary vertebral (intraossous) hemangioma with bilateral extension through both intervetebral foramen into the epidural space causing myelopathy.
To validate the English version of the Spielberger State-Trait Anxiety Inventory (STAI) in a sample of Malaysia patients with and without urinary symptoms. Validity and reliability were studied in patients with lower urinary tract symptoms (LUTS) and patients without LUTS. Reliability was evaluated using the test-retest method and internal consistency was assessed using Cronbach's alpha. Sensitivity to change was expressed as the effect size in the pre-intervention versus post-intervention score in additional patients with LUTS who underwent transurethral resection of the prostate (TURP). Internal consistency was excellent. A high degree of internal consistency was observed for each of the 40 items with Cronbach's alpha value = 0.38 to 0.89 while the Cronbach's alpha for the total scores was 0.86. Test-retest correlation coefficients for the 40 items score were highly significant. Intraclass correlation coefficient was high (ICC=0.39 to 0.89). A high degree of sensitivity and specificity to the effects of treatment was observed. A high degree of significant level between baseline and post-treatment scores was observed across nearly half of the items in surgical group but not in the non-LUTS group (control subjects). The STAI is reliable, valid and sensitive to clinical change in a sample of Malaysian patients with and without urinary symptoms.
Creating novel nanostructures is a primary step for high-performance analytical sensing. Herein, a new worm like nanostructure with Zinc Oxide-gold (ZnO/Au) hybrid was fabricated through an aqueous hydrothermal method, by doping Au-nanoparticle (AuNP) on the growing ZnO lattice. During ZnO growth, fine tuning the solution temperature expedites random curving of ZnO nanorods and forms nano-worms. The nano-worms which were evidenced by morphological, physical and structural analyses, revealed elongated structures protruding from the surface (length: 1 µm; diameter: ~100 nm). The appropriate peaks for the face centred cubic gold were (111) and (200), as seen from X-ray diffractogram. The strong interrelation between Au and ZnO was manifested by X-ray photoelectron spectroscopy. The combined surface area increment from the nanoparticle radii and ZnO nanorod random curving gives raise an enhancement in detection sensitivity by increasing bio-loading. 'Au-decorated hybrid nano-worm' was immobilized with a probe DNA from Vibrio Cholera and duplexed with a target which was revealed by Fourier Transform Infrared Spectroscopy. Our novel Au-decorated hybrid nano-worm is suitable for high-performance bio-sensing, as evidenced by impedance spectroscopy, having higher-specificity and attained femtomolar (10 fM) sensitivity. Further, higher stability, reproducibility and regeneration on this sensing surface were demonstrated.
Rationally designed biosensing system supports multiplex analyses is warranted for medical diagnosis to determine the level of analyte interaction. The chemically functionalized novel multi-electrode polysilicon nanogap (PSNG) lab-on-chip is designed in this study, facilitates multiplex analyses for a single analyte. On the fabricated 69nm PSNG, biocompatibility and structural characteristics were verified for the efficient binding of Human Chorionic Gonadotropin (hCG). With the assistance of microfluidics, hCG sample was delivered via single-injection to 3-Aminopropyl(triethoxy)silane (APTES) and Glycidoxypropyl(trimethoxy)silane (GPMS) modified PSNG electrodes and the transduced signal was used to investigate the dielectric mechanisms for multiplex analyses. The results from amperometric response and impedance measurement delivered the scale of interaction between anti-hCG antibody and hCG that exhibited 6.5 times higher sensitivity for the chemical linker, APTES than GPMS. Under optimized experimental conditions, APTES and GPMS modified immunosensor has a limit of detection as 0.56mIU/ml and 2.93mIU/ml (at S/N=3), with dissociation constants (Kd) of 5.65±2.5mIU/ml and 7.28±2.6mIU/ml, respectively. These results suggest that multiplex analysis of single target could enhance the accuracy of detection and reliable for real-time comparative analyses. The designed PSNG is simple, feasible, requires low sample consumption and could be applied for any given multiplex analyses.
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.
In the current study, facile synthesis of carboxymethyl cellulose (CMC) and sodium alginate capped silver nanoparticles (AgNPs) was examined using microwave radiation and aniline as a reducing agent. The biopolymer matrix embedded nanoparticles were synthesized under various experimental conditions using different concentrations of biopolymer (0.5, 1, 1.5, 2%), volumes of reducing agent (50, 100, 150 μL), and duration of heat treatment (30 s to 240 s). The synthesized nanoparticles were analyzed by scanning electron microscopy, UV-Vis spectroscopy, X-ray diffraction, and Fourier transform infrared spectroscopy for identification of AgNPs synthesis, crystal nature, shape, size, and type of capping action. In addition, the significant antibacterial efficacy and antibiofilm activity of biopolymer capped AgNPs were demonstrated against different bacterial strains, Staphylococcus aureus MTCC 740 and Escherichia coli MTCC 9492. These results confirmed the potential for production of biopolymer capped AgNPs grown under microwave irradiation, which can be used for industrial and biomedical applications.
Breast cancer is one of the most common cancers that affect women globally and accounts for ~23% of all cancers diagnosed in women. Breast cancer is also one of the leading causes of death primarily due to late stage diagnoses and a lack of effective treatments. Therefore, discovering protein expression biomarkers is mandatory for early detection and thus, critical for successful therapy. Two-dimensional electrophoresis (2D-E) coupled with lectin-based analysis followed by mass spectrometry were applied to identify potential biomarkers in the secretions of a murine mammary carcinoma cell line. Comparisons of the protein profiles of the murine 4T1 mammary carcinoma cell line and a normal murine MM3MG mammary cell line indicated that cadherin-1 (CDH), collagenase 3 (MMP-13), Viral envelope protein G7e (VEP), Gag protein (GAG) and Hypothetical protein LOC433182 (LOC) were uniquely expressed by the 4T1 cells, and pigment epithelium-derived factor (PEDF) was exclusively secreted by the MM3MG cells. Further analysis by a lectin-based study revealed that aberrant O-glycosylated CDH, N-glycosylated MMP-13 and LOC were present in the 4T1 medium. These differentially expressed N- and O-linked glycoprotein candidates, which were identified by combining lectin-based analysis with 2D-E, could serve as potential diagnostic and prognostic markers for breast cancer.
Diabetes is a metabolic disease with a prolonged elevated level of glucose in the blood leads to long-term complications and increases the chances for cardiovascular diseases. The present study describes the fabrication of a ZnO nanowire (NW)-modified interdigitated electrode (IDE) to monitor the level of blood glucose. A silver IDE was generated by wet etching-assisted conventional lithography, with a gap between adjacent electrodes of 98.80 μm. The ZnO-based thin films and NWs were amended by sol-gel and hydrothermal routes. High-quality crystalline and c-axis orientated ZnO thin films were observed by XRD analyses. The ZnO thin film was annealed for 1, 3 and 5 h, yielding a good-quality crystallite with sizes of 50, 100 and 110 nm, and the band gaps were measured as 3.26, 3.20 and 3.17 eV, respectively. Furthermore, a flower-modeled NW was obtained with the lowest diameter of 21 nm. Our designed ZnO NW-modified IDE was shown to have a detection limit as low as 0.03 mg/dL (correlation coefficient = 0.98952) of glucose with a low response time of 3 s, perform better than commercial glucose meter, suitable to instantly monitor the glucose level of diabetes patients. This study demonstrated the high performance of NW-mediated IDEs for glucose sensing as alternative to current glucose sensors.
Aptamers are single-stranded oligonucleotides that can be artificially generated by a method called Systematic evolution of ligands by exponential enrichment (SELEX). The generated aptamers have been assessed for high-performance sensing applications due to their appealing characteristics. With either aptamers alone or complementing with antibodies, several high sensitive and portable sensors have been demonstrated for use in 'point-of-care testing'. Due to their high suitability and flexibility, aptamers are conjugated with nanostructures and utilized in field applications. Moreover, aptamers are more amenable to chemical modifications, making them capable of utilization with most developed sensors. In this overview, we discuss novel, portable, and aptamer-based sensing strategies that are suitable for 'point-of-care testing'.
Breast cancer is one of the major issues in the field of oncology, reported with a higher prevalence rate in women worldwide. In attempt to reveal the potential biomarkers for breast cancer, the findings of differentially glycosylated haptoglobin and osteonectin in previous study have drawn our attention towards glycoproteins of secretome from the MCF-7 cancer cell line. In the present study, further analyses were performed on the medium of MCF-7 cells by subjecting it to two-dimensional analyses followed by image analysis in contrast to the medium of human mammary epithelial cells (HMEpC) as a negative control. Carboxypeptidase A4 (CPA4), alpha-1-antitrypsin (AAT), haptoglobin (HP), and HSC70 were detected in the medium of MCF-7, while only CPA4 and osteonectin (ON) were detected in HMEpC medium. In addition, CPA4 was detected as upregulated in the MCF-7 medium. Further analysis by lectin showed that CPA4, AAT, HP, and HSC70 were secreted as N-glycan in the medium of MCF-7, with HP also showing differentially N-glycosylated isoforms. For the HMEpC, only CPA4 was detected as N-glycan. No O-glycan was detected in the medium of HMEpC but MCF-7 expressed O-glycosylated CPA4 and HSC70. All these revealed that glycoproteins could be used as glycan-based biomarkers for the prognosis of breast cancer.
The performance of sensing surfaces highly relies on nanostructures to enhance their sensitivity and specificity. Herein, nanostructured zinc oxide (ZnO) thin films of various thicknesses were coated on glass and p-type silicon substrates using a sol-gel spin-coating technique. The deposited films were characterized for morphological, structural, and optoelectronic properties by high-resolution measurements. X-ray diffraction analyses revealed that the deposited films have a c-axis orientation and display peaks that refer to ZnO, which exhibits a hexagonal structure with a preferable plane orientation (002). The thicknesses of ZnO thin films prepared using 1, 3, 5, and 7 cycles were measured to be 40, 60, 100, and 200 nm, respectively. The increment in grain size of the thin film from 21 to 52 nm was noticed, when its thickness was increased from 40 to 200 nm, whereas the band gap value decreased from 3.282 to 3.268 eV. Band gap value of ZnO thin film with thickness of 200 nm at pH ranging from 2 to 10 reduces from 3.263eV to 3.200 eV. Furthermore, to evaluate the transducing capacity of the ZnO nanostructure, the refractive index, optoelectric constant, and bulk modulus were analyzed and correlated. The highest thickness (200 nm) of ZnO film, embedded with an interdigitated electrode that behaves as a pH-sensing electrode, could sense pH variations in the range of 2-10. It showed a highly sensitive response of 444 μAmM-1cm-2 with a linear regression of R2 =0.9304. The measured sensitivity of the developed device for pH per unit is 3.72μA/pH.
Lipases are of great interest for different industrial applications due to their diversity and versatility. Among different lipases, microbial lipases are preferable due to their broad substrate specificity, and higher stability with lower production costs compared to the lipases from plants and animals. In the past, a vast number of bacterial species have been reported as potential lipases producers. In this study, the lipases-producing bacterial species were isolated from an oil spillage area in the conventional night market. Isolated species were identified as Bacillus species by biochemical tests which indicate their predominant establishment, and further screened on the agar solid surfaces using lipid and gelatin as the substrates. Out of the ten strains tested, four potential strains were subjected to comparison analysis of the lipolytic versus proteolytic activities. Strain 10 exhibited the highest lipolytic and proteolytic activity. In all the strains, the proteolytic activity is higher than the lipolytic activity except for strain 8, suggesting the possibility for substrate-based extracellular gene induction. The simultaneous secretion of both the lipase and protease is a mean of survival. The isolated bacterial species which harbour both lipase and protease enzymes could render potential industrial-based applications and solve environmental issues.
Hybrid gold nanostructures seeded into nanotextured zinc oxide (ZnO) nanoflowers (NFs) were created for novel biosensing applications. The selected 'spotted NFs' had a 30-nm-thick gold nanoparticle (AuNP) layer, chosen from a range of AuNP thicknesses, sputtered onto the surface. The generated nanohybrids, characterized by morphological, physical and structural analyses, were uniformly AuNP-seeded onto the ZnO NFs with an average length of 2-3 μm. Selective capture of molecular probes onto the seeded AuNPs was evidence for the specific interaction with DNA from pathogenic Leptospirosis-causing strains via hybridization and mis-match analyses. The attained detection limit was 100 fM as determined via impedance spectroscopy. High levels of stability, reproducibility and regeneration of the sensor were obtained. Selective DNA immobilization and hybridization were confirmed by nitrogen and phosphorus peaks in an X-ray photoelectron spectroscopy analysis. The created nanostructure hybrids illuminate the mechanism of generating multiple-target, high-performance detection on a single NF platform, which opens a new avenue for array-based medical diagnostics.
The aim of the study was to validate the Malay version of the General Quentionnaire (GHQ-12) in patients with psychiatric morbidity secondary to urological disorder. Validity and reliability were studied in patients with lower urinary tract symptoms (LUTS) and patients without LUTS. Internal consistency was excellent. A high degree of internal consistency was observed for each of the 12 items and total scores (Cronbach's alpha value = 0.50 and higher and 0.65 respectively. Test-retest correlation coefficient for the 12 items scores was highly significant. Intraclass correlation coefficient was high (ICC=0.47 and above). A significant level between baseline and post-treatment scores were observed across 3 items in the surgical group. The Mal-GHQ-12 is a suitable, reliable, valid and sensitive to clinical change in the Malaysian population.