Displaying publications 1 - 20 of 40 in total

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  1. A rapid and cost effective method in purifying small RNA
    Citartan M, Tan SC, Tang TH
    World J Microbiol Biotechnol, 2012 Jan;28(1):105-11.
    PMID: 22806785 DOI: 10.1007/s11274-011-0797-0
    Purification of RNA fragments from a complex mixture is a very common technique, and requires consideration of the time, cost, purity and yield of the purified RNA fragments. This study describes the fastest method of purifying small RNA with the lowest cost possible, without compromizing the yield and purity. The technique describes the purification of small RNA from polyacrylamide gel, resulting in a good yield of small RNA with minimum experimental steps in avoiding degradation of the RNA, obviating the use of ethidium bromide and phenol-chloroform extraction, as well as siliconized glass wools to remove the polyacrylamide gel particles. The purified small RNA is suitable for a wide variety of applications such as ligation, end labelling with radio isotope, RT-PCR (Reverse Transcriptase-PCR), Northern blotting, experimental RNomics study and also Systematic Evolution of Ligands by Exponential Enrichment (SELEX).
  2. Assays for aptamer-based platforms
    Citartan M, Gopinath SC, Tominaga J, Tan SC, Tang TH
    Biosens Bioelectron, 2012 Apr 15;34(1):1-11.
    PMID: 22326894 DOI: 10.1016/j.bios.2012.01.002
    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.
  3. Label-free methods of reporting biomolecular interactions by optical biosensors
    Citartan M, Gopinath SC, Tominaga J, Tang TH
    Analyst, 2013 Jul 7;138(13):3576-92.
    PMID: 23646346 DOI: 10.1039/c3an36828a
    Reporting biomolecular interactions has become part and parcel of many applications of science towards an in-depth understanding of disease and gene regulation. Apart from that, in diagnostic applications where biomolecules (antibodies and aptamers) are vastly applied, meticulous monitoring of biomolecular interaction is vital for clear-cut diagnosis. Several currently available methods of analyzing the interaction of the ligands with the appropriate analytes are aided by labeling using fluorescence or luminescence techniques. However, labeling is cumbersome and can occupy important binding sites of interactive molecules to be labeled, which may interfere with the conformational changes of the molecules and increase non-specificity. Optical-based sensing can provide an alternative way as a label-free procedure for monitoring biomolecular interactions. Optical sensors affiliated with different operating principles, including surface plasmon changes, scattering and interferometry, can impart a huge impact for in-house and point-of-care applications. This optical-based biosensing permits real-time monitoring, obviating the use of hazardous labeling molecules such as radioactive tags. Herein, label-free ways of reporting biomolecular interactions by various optical biosensors were gleaned.
  4. Current aspects in immunosensors
    Gopinath SC, Tang TH, Citartan M, Chen Y, Lakshmipriya T
    Biosens Bioelectron, 2014 Jul 15;57:292-302.
    PMID: 24607580 DOI: 10.1016/j.bios.2014.02.029
    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.
  5. Use of UV-vis-NIR spectroscopy to monitor label-free interaction between molecular recognition elements and erythropoietin on a gold-coated polycarbonate platform
    Citartan M, Gopinath SC, Tominaga J, Chen Y, Tang TH
    Talanta, 2014 Aug;126:103-9.
    PMID: 24881539 DOI: 10.1016/j.talanta.2014.03.043
    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.
  6. Bacterial detection: from microscope to smartphone
    Gopinath SC, Tang TH, Chen Y, Citartan M, Lakshmipriya T
    Biosens Bioelectron, 2014 Oct 15;60:332-42.
    PMID: 24836016 DOI: 10.1016/j.bios.2014.04.014
    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.
  7. Sensing strategies for influenza surveillance
    Gopinath SC, Tang TH, Chen Y, Citartan M, Tominaga J, Lakshmipriya T
    Biosens Bioelectron, 2014 Nov 15;61:357-69.
    PMID: 24912036 DOI: 10.1016/j.bios.2014.05.024
    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.
  8. Monitoring recombinant human erythropoietin abuse among athletes
    Citartan M, Gopinath SCB, Chen Y, Lakshmipriya T, Tang TH
    Biosens Bioelectron, 2015 Jan 15;63:86-98.
    PMID: 25058943 DOI: 10.1016/j.bios.2014.06.068
    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.
  9. Aptamers as a replacement for antibodies in enzyme-linked immunosorbent assay
    Toh SY, Citartan M, Gopinath SC, Tang TH
    Biosens Bioelectron, 2015 Feb 15;64:392-403.
    PMID: 25278480 DOI: 10.1016/j.bios.2014.09.026
    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.
  10. Fulltext Microbial Enzymes and Their Applications in Industries and Medicine 2014
    Anbu P, Gopinath SC, Chaulagain BP, Tang TH, Citartan M
    Biomed Res Int, 2015;2015:816419.
    PMID: 26161416 DOI: 10.1155/2015/816419
  11. Fulltext Lipase-Secreting Bacillus Species in an Oil-Contaminated Habitat: Promising Strains to Alleviate Oil Pollution
    Lee LP, Karbul HM, Citartan M, Gopinath SC, Lakshmipriya T, Tang TH
    Biomed Res Int, 2015;2015:820575.
    PMID: 26180812 DOI: 10.1155/2015/820575
    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.
  12. Fulltext Non-protein coding RNA genes as the novel diagnostic markers for the discrimination of Salmonella species using PCR
    Nithya R, Ahmed SA, Hoe CH, Gopinath SC, Citartan M, Chinni SV, et al.
    PLoS One, 2015;10(3):e0118668.
    PMID: 25774907 DOI: 10.1371/journal.pone.0118668
    Salmonellosis, a communicable disease caused by members of the Salmonella species, transmitted to humans through contaminated food or water. It is of paramount importance, to generate accurate detection methods for discriminating the various Salmonella species that cause severe infection in humans, including S. Typhi and S. Paratyphi A. Here, we formulated a strategy of detection and differentiation of salmonellosis by a multiplex polymerase chain reaction assay using S. Typhi non-protein coding RNA (sRNA) genes. With the designed sequences that specifically detect sRNA genes from S. Typhi and S. Paratyphi A, a detection limit of up to 10 pg was achieved. Moreover, in a stool-seeding experiment with S. Typhi and S. Paratyphi A, we have attained a respective detection limit of 15 and 1.5 CFU/mL. The designed strategy using sRNA genes shown here is comparatively sensitive and specific, suitable for clinical diagnosis and disease surveillance, and sRNAs represent an excellent molecular target for infectious disease.
  13. Aptahistochemistry in diagnostic pathology: technical scrutiny and feasibility
    Bukari BA, Citartan M, Ch'ng ES, Bilibana MP, Rozhdestvensky T, Tang TH
    Histochem Cell Biol, 2017 May;147(5):545-553.
    PMID: 28321500 DOI: 10.1007/s00418-017-1561-9
    Antibodies have been the workhorse for diagnostic immunohistochemistry to specifically interrogate the expression of certain protein to aid in histopathological diagnosis. This review introduces another dimension of histochemistry that employs aptamers as the core tool, the so-called aptahistochemistry. Aptamers are an emerging class of molecular recognition elements that could recapitulate the roles of antibodies. The many advantageous properties of aptamers suited for this diagnostic platform are scrutinized. An in-depth discussion on the technical aspects of aptahistochemistry is provided with close step-by-step comparison to the more familiarized immunohistochemical procedures, namely functionalization of the aptamer as a probe, antigen retrieval, optimization with emphasis on incubation parameters and visualization methods. This review offers rationales to overcome the anticipated challenges in transition from immunohistochemistry to aptahistochemistry, which is deemed feasible for an average diagnostic pathology laboratory.
  14. Fulltext Current and Potential Developments of Cortisol Aptasensing towards Point-of-Care Diagnostics (POTC)
    Zainol Abidin AS, Rahim RA, Md Arshad MK, Fatin Nabilah MF, Voon CH, Tang TH, et al.
    Sensors (Basel), 2017 May 22;17(5).
    PMID: 28531146 DOI: 10.3390/s17051180
    Anxiety is a psychological problem that often emerges during the normal course of human life. The detection of anxiety often involves a physical exam and a self-reporting questionnaire. However, these approaches have limitations, as the data might lack reliability and consistency upon application to the same population over time. Furthermore, there might be varying understanding and interpretations of the particular question by the participant, which necessitating the approach of using biomarker-based measurement for stress diagnosis. The most prominent biomarker related to stress, hormone cortisol, plays a key role in the fight-or-flight situation, alters the immune response, and suppresses the digestive and the reproductive systems. We have taken the endeavour to review the available aptamer-based biosensor (aptasensor) for cortisol detection. The potential point-of-care diagnostic strategies that could be harnessed for the aptasensing of cortisol were also envisaged.
  15. Fulltext Aptamers as the Agent in Decontamination Assays (Apta-Decontamination Assays): From the Environment to the Potential Application In Vivo
    Bilibana MP, Citartan M, Yeoh TS, Rozhdestvensky TS, Tang TH
    J Nucleic Acids, 2017;2017:3712070.
    PMID: 29225967 DOI: 10.1155/2017/3712070
    The binding specificity and affinity of aptamers have long been harnessed as the key elements in the development of aptamer-based assays, particularly aptasensing application. One promising avenue that is currently explored based on the specificity and affinity of aptamers is the application of aptamers in the decontamination assays. Aptamers have been successfully harnessed as the decontamination agents to remove contaminants from the environment and to decontaminate infectious elements. The reversible denaturation property inherent in aptamers enables the repeated usage of aptamers, which can immensely save the cost of decontamination. Analogous to the point-of-care diagnostics, there is no doubt that aptamers can also be deployed in the point-of-care aptamer-based decontamination assay, whereby decontamination can be performed anywhere and anytime for instantaneous decision-making. It is also prophesied that aptamers can also serve more than as a decontaminant, probably as a tool to capture and kill hazardous elements, particularly pathogenic agents.
  16. Bacterial regulatory RNAs: complexity, function, and putative drug targeting
    Cheah HL, Raabe CA, Lee LP, Rozhdestvensky TS, Citartan M, Ahmed SA, et al.
    Crit Rev Biochem Mol Biol, 2018 08;53(4):335-355.
    PMID: 29793351 DOI: 10.1080/10409238.2018.1473330
    Over the past decade, RNA-deep sequencing has uncovered copious non-protein coding RNAs (npcRNAs) in bacteria. Many of them are key players in the regulation of gene expression, taking part in various regulatory circuits, such as metabolic responses to different environmental stresses, virulence, antibiotic resistance, and host-pathogen interactions. This has contributed to the high adaptability of bacteria to changing or even hostile environments. Their mechanisms include the regulation of transcriptional termination, modulation of translation, and alteration of messenger RNA (mRNA) stability, as well as protein sequestration. Here, the mechanisms of gene expression by regulatory bacterial npcRNAs are comprehensively reviewed and supplemented with well-characterized examples. This class of molecules and their mechanisms of action might be useful targets for the development of novel antibiotics.
  17. In silico 'fishing' using known small regulatory RNA (sRNA) candidates as the decoy from Escherichia coli, Salmonella typhi and Salmonella typhimurium manifested 14 novel sRNA candidates in the orthologous region of Proteus mirabilis
    KishanRaj S, Sumitha S, Siventhiran B, Thiviyaa O, Sathasivam KV, Xavier R, et al.
    Mol Biol Rep, 2018 Dec;45(6):2333-2343.
    PMID: 30284142 DOI: 10.1007/s11033-018-4397-z
    Proteus mirabilis, a gram-negative bacterium of the family Enterobacteriaceae, is a leading cause of urinary tract infection (UTI) with rapid development of multi-drug resistance. Identification of small regulatory RNAs (sRNAs), which belongs to a class of RNAs that do not translate into a protein, could permit the comprehension of the regulatory roles this molecules play in mediating pathogenesis and multi-drug resistance of the organism. In this study, comparative sRNA analysis across three different members of Enterobacteriaceae (Escherichia coli, Salmonella typhi and Salmonella typhimurium) was carried out to identify the sRNA homologs in P. mirabilis. A total of 232 sRNA genes that were reported in E. coli, S. typhi and S. typhimurium were subjected to comparative analysis against P. mirabilis HI4320 genome. We report the detection of 14 sRNA candidates, conserved in the orthologous regions of P. mirabilis, that are not included in Rfam database. Northern-blot analysis was carried out for selected three sRNA candidates from the current investigation and three known sRNA from Rfam of P. mirabilis. The expression pattern of the six sRNA candidates shows that they are growth stage-dependant. To the best of our knowledge, this is the first report on the identification of sRNA candidates in P. mirabilis.
  18. A simple method for in-house Pfu DNA polymerase purification for high-fidelity PCR amplification
    Sankar PS, Citartan M, Siti AA, Skryabin BV, Rozhdestvensky TS, Khor GH, et al.
    Iran J Microbiol, 2019 Apr;11(2):181-186.
    PMID: 31341574
    Background and Objectives: Pfu DNA polymerase is an enzyme that exhibits the lowest error rate in the 3' to 5' exonuclease (proofreading) activity during DNA synthesis in Polymerase Chain Reactions (PCRs). This study was aimed to express and purify Pfu DNA polymerase in a bacterial expression system under a simple purification method.

    Materials and Methods: Pfu polymerase gene sequence, derived from Pyrocuccus furiosus (Pfu) genomic DNA, was cloned and overexpressed in E. coli BL21 (DE3) pLysS. Upon overexpression, bacterial lysate containing the Pfu DNA polymerase was heated at 94°C for 5 minutes. Pfu DNA polymerase having high thermal stability was retained while the other bacterial proteins were denatured. The resulting thermo stable Pfu DNA polymerase was separated from the other debris of the denatured proteins by simple centrifugation.

    Results: The enzymatic activity of the resulting Pfu DNA polymerase was estimated by comparing with the commercial Pfu DNA Polymerases. An estimated 50000 units of functional Pfu DNA polymerase was produced from a 400 ml culture.

    Conclusion: The in-house produced Pfu DNA Polymerase could be used for routine amplification that requires high-fidelity such as cloning and DNA sequencing.

  19. Recent developments of aptasensors expedient for point-of-care (POC) diagnostics
    Citartan M, Tang TH
    Talanta, 2019 Jul 01;199:556-566.
    PMID: 30952298 DOI: 10.1016/j.talanta.2019.02.066
    Aptamers are nucleic acid-based molecular recognition elements that are specific and have high binding affinity against their respective targets. On account of their target recognition capacity, aptamers are widely utilized in a number of applications including diagnostics. This review aims to highlight the recent developments of aptasensors expedient for point-of-care (POC) diagnostics. Significant focus is given on the primary assay formats of aptamers such as fluorescence, electrochemical, surface plasmon resonance (SPR) and colorimetric assays. A potpourri of platforms such as paper-based device, lateral flow assay, portable electrodes, portable SPR and smart phones expedient for point-of-care (POC) diagnostics are discussed. Emphasis is also given on the technicalities and assay configurations associated with the sensors.
  20. Aptamers as the chaperones (Aptachaperones) of drugs-from siRNAs to DNA nanorobots
    Citartan M, Kaur H, Presela R, Tang TH
    Int J Pharm, 2019 Aug 15;567:118483.
    PMID: 31260780 DOI: 10.1016/j.ijpharm.2019.118483
    Aptamers, nucleic acid ligands that are specific against their corresponding targets are increasingly employed in a variety of applications including diagnostics and therapeutics. The specificity of the aptamers against their targets is also used as the basis for the formulation of the aptamer-based drug delivery system. In this review, we aim to provide an overview on the chaperoning roles of aptamers in acting as the cargo or load carriers, delivering contents to the targeted sites via cell surface receptors. Internalization of the aptamer-biomolecule conjugates via receptor-mediated endocytosis and the strategies to augment the rate of endocytosis are underscored. The cargos chaperoned by aptamers, ranging from siRNAs to DNA origami are illuminated. Possible impediments to the aptamer-based drug deliveries such as susceptibility to nuclease resistance, potentiality for immunogenicity activation, tumor heterogeneity are speculated and the corresponding amendment strategies to address these shortcomings are discussed. We prophesy that the future of the aptamer-based drug delivery will take a trajectory towards DNA nanorobot-based assay.
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