|Year : 2023 | Volume
| Issue : 3 | Page : 119-128
Tapered optical fiber DNA biosensor for detecting Leptospira DNA
Jia-Yong Lam1, Mohd Hanif Yaacob2, Hui-Yee Chee3
1 Department of Medical Microbiology, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia
2 Department of Computer and Communication Systems Engineering, Faculty of Engineering; Wireless and Photonics Networks Research Centre (WiPNET), Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia
3 Department of Medical Microbiology, Faculty of Medicine and Health Sciences; Wireless and Photonics Networks Research Centre (WiPNET), Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia
|Date of Submission||05-Nov-2022|
|Date of Decision||08-Feb-2023|
|Date of Acceptance||10-Feb-2023|
|Date of Web Publication||28-Mar-2023|
Department of Medical Microbiology, Faculty of Medicine and Health Sciences; Wireless and Photonics Networks Research Centre (WiPNET), Universiti Putra Malaysia, 43400 UPM Serdang, Selangor
Source of Support: This research was funded by Universiti Putra Malaysia through the Geran Inisiatif Putra Siswazah (GP-IPS/2019/9678200), Conflict of Interest: None
Objective: To establish a DNA detection platform based on a tapered optical fiber to detect Leptospira DNA by targeting the leptospiral secY gene.
Methods: The biosensor works on the principle of light propagating in the special geometry of the optical fiber tapered from a waist diameter of 125 to 12 µm. The fiber surface was functionalized through a cascade of chemical treatments and the immobilization of a DNA capture probe targeting the secY gene. The presence of the target DNA was determined from the wavelength shift in the optical transmission spectrum.
Results: The biosensor demonstrated good sensitivity, detecting Leptospira DNA at 0.001 ng/µL, and was selective for Leptospira DNA without cross-reactivity with non-leptospiral microorganisms. The biosensor specifically detected DNA that was specifically amplified through the loop-mediated isothermal amplification approach.
Conclusions: These findings warrant the potential of this platform to be developed as a novel alternative approach to diagnose leptospirosis.
Keywords: DNA biosensor; Tapered optical fiber; Leptospirosis; Leptospira
|How to cite this article:|
Lam JY, Yaacob MH, Chee HY. Tapered optical fiber DNA biosensor for detecting Leptospira DNA. Asian Pac J Trop Med 2023;16:119-28
| 1. Introduction|| |
Climate change and recurrent natural disasters have favored pathogens in the environment, causing the emergence of waterborne and vector-borne diseases, such as leptospirosis. Leptospirosis is caused by spirochetes in the Leptospira genus. Early diagnosis of leptospirosis is crucial for prompt intervention, as the therapeutic strategies are most effective when initiated during the early phase. However, the nonspecific clinical features during the early acute phase often complicate the diagnosis. The distinctive clinical manifestations of leptospirosis are only observed when the disease has worsened into the severe late phase. As such, leptospirosis is often the cause of undifferentiated febrile illness, particularly in regions where other infectious diseases with overlapping manifestations are endemic,. Without a robust diagnosis, identifying leptospirosis is often dependent on the suspicion of the clinician but presumptive treatment is common for managing the disease.
The gold-standard tests, such as culture and the microscopic agglutination test, are laborious and require proper quality controls; hence, they are often ineffective for an early rapid diagnosis of the disease. Given the potential severity of the disease, molecular detection, such as a DNA-based approach, is a more effective option to detect the etiological agent [4,10]. Detection based on DNA results in higher sensitivity and specificity than the gold-standard or serological tests, particularly during the early phase of the disease. Nucleic acids have been extensively applied in a wide range of biosensors due to their versatile physical, chemical, and biological activities. The biological recognition element in a biosensor, or the bioreceptor, is immobilized on a physical transducer to convert the bioreceptor interaction with its respective analyte into a measurable signal. Hence, the recognition of the bioreceptor for the analyte is based on highly specific complementary binding of nucleotides, allowing for sequence-specific information in the form of measurable signals.
Optical fibers have become an important part of biosensing technology due to their compact size and protection from electromagnetic interference,. They are often used as transduction elements in biosensors, relying on certain optical transduction mechanisms for detecting the target analyte. In particular, tapered optical fibers have been extensively used to develop systems to detect various biomolecules,. Unlike a typical optical fiber, where light propagation is confined inside the core with minimal loss and decay in the cladding where reflection occurs, the tapering process of a tapered optical fiber allows exposure to the evanescent field. This design allows for an interaction between the transmitted light and the external medium, which, in turn, allows for the evanescent sensing mechanism on the fiber. In DNA biosensing, the evanescent wave interacts with DNA molecules along the sensitive tapered distance of the fiber, causing a change in frequency, phase, or intensity of light with respect to the quantity and configuration of the DNA molecules,,. The mathematical principle of biosensing using a tapered optical fiber has been described in a previous study and suggested that changing the refractive index at the external surroundings of the tapered region would affect the phase shift, fringe shift, and output intensity of the light.
In this study, a DNA detection platform was developed based on a tapered optical fiber to detect Leptospira spp. The selectivity of the biosensor toward Leptospira was ensured by using a DNA probe immobilized on the surface of the fiber that specifically targeted the secY gene, a housekeeping gene present in all Leptospira spp. The biosensor demonstrated a concentration-dependent response upon exposure to Leptospira genomic DNA and was capable of detecting a low concentration of Leptospira genomic DNA.
| 2. Materials and methods|| |
2.1. DNA probe design
The secY gene sequences from various Leptospira serovars (Serovars: Pomona, Canicola, Bataviae, Icterohaemorrhagiae, Hardjo-prajitno, Lai, Djasiman, Autumnalis; Accession numbers: EU357943.1, EU357947.1, EU357956.1, EU357961.1, EU357983.1, EU357997.1, EU358012.1, and EU358013.1), as well as non-Leptospira microorganisms [Vibrio cholerae, Escherichia (E.) coli O157: H7, Salmonella enterica, Campylobacter jejuni, Clostridioides (C.) difficile; Accession numbers: NZ_CP043554.1, NC_002695.2, NC_003197.2, NC_002163.1, and NZ_CP076401.1] were retrieved from the NCBI nucleotide database (https://www.ncbi.nlm.nih.gov/nucleotide/). Multiple sequence alignment was performed on the sequences using Clustal Omega software (https://www.ebi.ac.uk/Tools/msa/clustalo/) to identify the conserved region of the secY gene. A DNA probe with the sequence 5'-CTT GTT CCT GCC CTT CAA A-3' was designed and synthesized based on the alignment data (Integrated DNA Technologies, Coralville, IA, USA).
2.2. Extraction of the genomic DNA
Genomic DNA of the Leptospira reference strains and other non-Leptospira bacteria were extracted from their respective pure cultures using the Wizard® Genomic DNA Purification Kit (Promega Corp., Madison, WI, USA) according to the manufacturer’s procedure. The extracted DNAs were quantified for concentration and purity using the NanoDrop™ ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), and stored at 20 °C until further use.
2.3. Fabrication of the tapered optical fiber and experimental setup
Single-mode optical fibers (SMFs) (Lucent Technologies, New Providence, NJ, USA) with a standard core and cladding diameters of ~8 µm and 125 µm, respectively, were utilized to fabricate the tapered optical fibers in this study. The tapered fibers were fabricated using the Vytran GPX-3400 glass processing workstation (Vytran, Morganville, NJ, USA) by modifying the optical fiber dimensions using the heat and pull principle. The real-time control system of the workstation enables users to control the dimensions and uniformity of the fibers, by ensuring that the pulling speed remains constant at 1 mm/s, and heat power at 42 W. The initial cladding diameter of the fiber was tapered down to 12 µm, with a symmetric up-and-down transition of the taper at the length of 5 mm, and a tapered waist length fixed at 15 mm. The dimensions of the tapered optical fiber were further validated using the machine’s microscope and camera. The dimensions of the tapered fibers used in this study have been optimized and reported in previous studies,.
The fabricated tapered optical fiber was secured onto a custom-made sample holder with the tapered region positioned within the straight sample well-groove. The reagents and analytes were introduced to the tapered region by pipetting into the vacant groove. Both ends of the optical fiber were spliced into SMF pigtails using an optical fiber fusion splicer (Sumitomo Electric, Osaka, Japan). The input end of the pigtail was connected to the single-mode FC/PC patch cord of the Amonics ALS 18-B-FA broadband light source (Amonics, San Po Kong, Hong Kong), while the output end was connected to the Yokogawa AQ6331 optical spectrum analyzer (OSA) (Yokogawa, Tokyo, Japan). The transmission output spectrum from the OSA was recorded into a computer using LabView in-house software.
2.4. Surface functionalization of the tapered optical fiber
The tapered region must undergo a series of functionalization steps to be immobilized with the DNA probe to capture leptospiral DNA. Functionalization is essential to promote the conjugation between the inorganic surface, such as the optical fiber, to the organic biological elements. Surface functionalization was initiated by incubating the tapered fiber in 0.1 M sodium hydroxide (NaOH) (Sigma-Aldrich, St. Louis, MO, USA) for 45 min. The solution was drained, and the tapered region was rinsed three times with deionized water. Then, 2% v/v (3-aminopropyl) triethoxysilane (APTES) (Sigma-Aldrich) was introduced to the tapered region and incubated for 45 min. The tapered region was rinsed three times with deionized water and left to air-dry at room temperature. A 2% v/v glutaraldehyde solution (VWR International, Monroeville, PA, USA) was applied to the tapered region for 1 hour, followed by the same rinsing and drying steps. A 1 µM probe solution was introduced onto the tapered region of the optical fiber and incubated for 1 hour to immobilize the DNA probe. The surface of the tapered region was rinsed three times with deionized water and air-dried at room temperature. The experimental setup of the biosensor detection system is illustrated in [Figure 1].
|Figure 1: Experimental setup of the tapered optical fiber biosensor and the detection system.|
Click here to view
2.5. Hybridization and detection of the target DNA
The transmission output spectrum of the immobilized DNA probe was recorded via OSA as “Probe” before introducing the DNA sample onto the tapered region of the fiber. The genomic DNA was heated at 95 °C for 5 min, followed by an immediate cold shock on ice for 1 min to denature the double-stranded DNA into single strands for DNA hybridization. The denatured DNA sample was pipetted onto the tapered region of the functionalized optical fiber and allowed to hybridize for 1 hour. After the incubation, the unbound DNA was aspirated off and the tapered region was rinsed three times with deionized water and left to air-dry at room temperature. The transmission output spectrum was once again recorded via the OSA as the “DNA sample.” The data were transferred to a spreadsheet for analysis, where the transmission spectra for the “Probe” and the “DNA sample” were compared and the wavelength shift in both spectra was determined.
2.6. Sensitivity and specificity testing of the tapered optical fiber biosensor
The genomic DNA isolated from Leptospira (L.) interrogans was serially diluted ten-fold from 0.1 to 0.001 ng/µL to determine the analytical sensitivity of the biosensor. The diluted genomic DNA was separately tested on each taper profile in triplicate. The genomic DNAs isolated from 17 Leptospira reference strains (pathogenic L. interrogans serovars Serawak, Canicola, Djasiman, Autumnalis, Australis, Pyrogenes, Lai, Copenhageni, Terengganu, Icterohaemorrhagiae, Bataviae, and Melaka; pathogenic L. borgpetersenii serovars Bataviae and Javanica; pathogenic L. kirschneri serovar Grippotyphosa; saprophytic L. biflexa serovar Patoc) and 3 non-Leptospira bacteria (C. difficile, E. coli strain XL10G, and a clinical isolate of methicillin-resistant Staphylococcus aureus) were used to assess the specificity of the biosensor, in which 0.1 ng/µL of each genomic DNA was tested in triplicate. All bacterial cultures were obtained from the Microbiology Laboratory, Universiti Putra Malaysia, Malaysia, where they were maintained. As described in a previous study, the Leptospira reference strains used by the Microbiology Laboratory, Universiti Putra Malaysia, Malaysia were obtained from the World Health Organization Leptospirosis Collaborating Center, Amsterdam (the Netherlands) and the Institute for Medical Research, Malaysia.
2.7. Detection of amplified DNA using the tapered optical fiber biosensor
The tapered optical fiber biosensor was tested for detecting the target DNA amplified using the loop-mediated isothermal amplification (LAMP) method. The LAMP assay was designed to target the Leptospira secY gene as described previously, in which the reaction parameters were optimized with slight modifications.
The LAMP reaction was assembled in a 25 µL reaction volume comprised of 1× Bst ThermoPol® buffer (New England Biolabs, New Ipswich, MA, USA), 6 mM magnesium sulfate, MgSO4 (New England Biolabs), 1.4 mM of each dNTP (First Base Laboratories, Selangor, Malaysia), 0.2 µM each of the F3 and B3 primers, 1.6 µM each of the FIP and BIP primers, 0.8 µM each of the LoopF and LoopB primers, and 8 U of Bst DNA polymerase, large fragment (New England Biolabs). Genomic DNA of L. interrogans serovar Pomona (3 and 30 ng) was used as the positive control template for the LAMP reactions. Sterile deionized water was used as the nontemplate control in place of the genomic DNA template. The LAMP reaction was performed for 20 min, and then the reaction was terminated at 85 °C for 5 min. The reaction products were analyzed by agarose gel electrophoresis, followed by ethidium bromide staining, and visualized on a gel documentation system (Bio-Rad Laboratories Inc., Hercules, CA, USA). A positive amplification reaction was characterized by the presence of a ladder-like pattern. The reaction products were maintained on ice before testing on the tapered optical fiber biosensor using the procedure described in Section 2.5.
| 3. Results|| |
3.1. Functionalization of the tapered optical fiber
The surface of the inorganic tapered optical fiber was modified to allow the binding of biological molecules, such as the DNA probe. [Figure 2]A depicts the surface functionalization process and the modifications made to the surface of the tapered fiber following a stepwise treatment with chemicals and linkers. [Figure 2]B shows the transmission output spectra of the bare tapered optical fiber and each of the surface functionalization steps. The transmission spectra demonstrated a red shift after each functionalization reaction. The spectrum shifted 2.11 nm to the right after NaOH hydroxylation. The spectrum continued a rightward shift of 1.68 nm and 0.87 nm following APTES silanization and the glutaraldehyde treatment, respectively. After the DNA probe was immobilized, the spectrum shifted another 1.36 nm, indicating that each of the steps altered the effective refractive index of the surface of the tapered optical fiber, suggesting successful chemical modification of the surface.
|Figure 2: Surface functionalization of the tapered optical fiber biosensor. (A) Schematic illustration of the geometry of the tapered optical fiber and its surface modification during functionalization. (B) Spectral response of the tapered optical fiber indicating a wavelength shift after each functionalization step from NaOH, APTES, glutaraldehyde, and DNA probe immobilization. APTES: (3-aminopropyl) triethoxysilane.|
Click here to view
3.2. Sensitivity of the tapered optical fiber biosensor
To determine analytical sensitivity, each dilution of the serially diluted L. interrogans genomic DNA (0.1-0.001 ng/µL) was separately tested on the functionalized tapered fiber biosensor. [Figure 3] shows the pre- and posthybridization transmission spectra for all three concentrations tested. C. difficile genomic DNA (0.1 ng/µL) was used as a nontarget control for comparison. The wavelength shift data for the sensitivity test are summarized in [Figure 4]. The biosensor demonstrated a concentration-dependent response to L. interrogans genomic DNA. A two-sample t-test revealed that the wavelength shift exhibited by the lowest concentration (0.001 ng/µL) of L. interrogans genomic DNA was significantly higher (P=0.000 2) than that of the nontarget C. difficile genomic DNA. The increase in the wavelength shift continued with the higher concentrations of L. interrogans genomic DNA with greater significance (P<0.000 1 for both 0.01 ng/µL and 0.1 ng/µL).
|Figure 3: Transmission output spectra of Leptospira interrogans genomic DNA at different concentrations. Transmission spectra when tested at (A) 0.1 ng/µL, (B) 0.01 ng/µL, and (C) 0.001 ng/µL of Leptospira interrogans genomic DNA.|
Click here to view
|Figure 4: Wavelength shift in the different concentrations of Leptospira interrogans genomic DNA on the tapered optical fiber biosensor. Unpaired two-sample t-tests were performed to compare the means of the wavelength shift for each tested concentration against the mean wavelength shift of the nontarget Clostridioides difficile. The P-value is indicated in the figure. Error bars indicate standard deviations.|
Click here to view
3.3. Specificity of the tapered optical fiber biosensor
The tapered optical fiber biosensor was tested for analytical specificity on 17 genomic DNAs from Leptospira reference strains and 3 from non-Leptospira bacteria. As shown in [Figure 5]A, the spectra pre- and posthybridization procedure of C. difficile DNA was comparable, with a minimal wavelength shift, indicating that hybridization did not occur between the probe and C. difficile DNA. Hybridization occurred in the presence of the target DNA, resulting in an altered fiber surface refractive index, thereby shifting the spectrum pattern, as shown in [Figure 5]B. The wavelength shifts for each of the genomic DNAs tested are quantitated in [Table 1]. The wavelength shifts produced by the Leptospira genomic DNAs were compared to the wavelength shifts of the nontarget controls (C. difficile, E. coli, and methicillin-resistant Staphylococcus aureus).
|Figure 5: Representative transmission output spectra of non-Leptospira and Leptospira genomic DNA. Transmission spectra when tested with 0.1 ng/µL of genomic DNA from (A) Clostridioides difficile and (B) the Leptospira interrogans serovar Canicola.|
Click here to view
|Table 1: Means of the wavelength shifts and statistical analysis of the non-target controls.|
Click here to view
The findings indicate that the biosensor significantly discriminated the 17 tested Leptospira genomic DNAs from the 3 non-Leptospira genomic DNAs (P<0.05), except the L. interrogans serovar Icterohaemorrhagiae compared to E. coli.
3.4. Detection of LAMP-amplified DNA using the tapered optical fiber biosensor
In the present study, the biosensor was also tested with LAMP-amplified DNA. The LAMP assay was performed on the nontemplate and positive controls under optimized conditions before detecting the respective wavelength shifts on the biosensor. [Figure 6]A shows the transmission spectra obtained when the biosensor was tested on the LAMP reaction products. The nontemplate control exhibited a minimal wavelength shift, which correlated with the absence of a ladder-like pattern on the agarose gel. The ladder-like pattern on the gel is characteristic of a positive DNA amplification reaction [Figure 6]B. As such, the wavelength shifts observed for the positive control (3 and 30 ng DNA template concentrations) correlated with the appearance of the ladder-like pattern signifying amplification of the target DNA, and the shifts were significant compared to the nontemplate control [Figure 6]C.
|Figure 6: Testing of the tapered optical fiber biosensor for detecting loop-mediated isothermal amplification (LAMP)-amplified target DNA. (A) Spectral response when tested on (from left to right) the LAMP reaction nontemplate control, LAMP amplicons from a 3 ng positive control template, and a 30 ng template. (B) The outcome of 1.5% agarose gel electrophoresis on the LAMP reaction products, where N represents the nontemplate control and lanes 1 and 2 represent the LAMP reaction products from the 3 and 30 ng positive control templates, respectively. (C) Wavelength shift in the LAMP reaction products when tested on the tapered optical fiber biosensor. Each bar represents the mean wavelength shift and error bars indicate standard deviations. The P-value from the unpaired two-sample t-tests is indicated in the figure.|
Click here to view
| 4. Discussion|| |
In the present study, a DNA biosensor based on a tapered optical fiber was developed to detect Leptospira DNA. The tapered optical fiber was functionalized through a cascade of chemical and linker treatments to allow the binding of the target analyte. The surface of the fiber was first modified with hydroxyl groups (-OH) by sodium hydroxide (NaOH) hydroxylation. These hydroxyl groups are vital for attaching the silanols for crosslinking reactions,. The silanes from APTES are affixed to the surface through the siloxane (Si-O-Si) linkage via the hydroxyl groups introduced earlier. The resulting surface is now modified with amine groups, which readily react with the bifunctional molecule glutaraldehyde. As such, a linker is formed to allow the covalent bonding of the DNA probe to the surface of the fiber. With the DNA probe in place, the target DNA is captured based on Watson-Crick base pair matching, and hybridized into a duplex formation.
In this study, the DNA probe was designed to target the leptospiral secY gene as opposed to the rrs gene probe used in a previous study. Located in the S10-spc-α locus containing genes for ribosomal proteins, the secY gene encodes the leptospiral preprotein translocase, a processive enzyme. The secY and rrs genes are Leptospira housekeeping genes and are ubiquitously expressed across all leptospiral species, thereby allowing the detection of pathogenic and saprophytic species of Leptospira,. The unusual presence of saprophytic Leptospira has been reportedly detected in suspected leptospirosis patients. As such, a detection platform that focuses only on the pathogenic strains may lead to false-negative results. A recent meta-analysis on the diagnostic accuracy of various leptospiral genetic markers, such as secY, rrs, lipL32, and flaB, revealed that nucleic acid-based assays targeting the secY gene are promising for detecting pathogens. Therefore, the secY gene was selected as the target gene in this work to enable extensive detection of leptospiral DNA regardless of its pathogenicity.
Although previous studies have successfully demonstrated the use of a tapered optical fiber as a DNA biosensing platform, synthesized short oligonucleotides were utilized as the complementary and non-complementary target DNA to determine the sensor’s sensitivity and specificity in these studies,,,. While using synthesized short oligonucleotides as target DNA allows flexibility to control the number of mismatches when testing a biosensor’s specificity, the downside is that it does not resemble the actual genome of the target organism, which is ultimately the analyte the biosensor was designed to capture. In a previous study targeting the rrs gene, the complementary and non-complementary oligonucleotides included 20 bases, resulting in a massive difference compared to the actual Leptospira genome size at approximately 5 Mb. Henceforth, genomic DNA isolated from a pure bacterial culture was utilized throughout this study to more accurately determine the sensitivity and specificity of the biosensor.
The biosensor demonstrated good sensitivity and detected Leptospira genomic DNA down to 0.001 ng/µL, which is lower than the reported limit of detection in a previous study targeting the rrs gene. In theory, due to the design of the DNA probe, only Leptospira DNA with the secY gene can be hybridized to the probe. Thus, when tested on a nontarget sample, such as C. difficile DNA, hybridization of the probe on the C. difficile DNA did not materialize, and hence caused no morphological or refractive index changes on the surface of the fiber, which was evident by the minimal wavelength shift observed. In contrast, when tested on the DNA of various Leptospira serovars, the biosensor differentiated these DNAs against the three nontarget controls, except one serovar. Technical limitations accounted for the variations in the wavelength shifts across experiments, which may have been contributed during the fabrication and surface functionalization process, as a new fiber was utilized for each test. In particular, the functionalization process is particularly susceptible to variability, as the chemical reactions involved are highly dependent on the temperature and humidity of the surroundings,.
The approach of combining a nucleic acid biosensor with a DNA amplification technique is intriguing due to the improved sensitivity and specificity of the biosensor,. Such a combination offers exciting opportunities to improve the development of integrated analytic systems, sensor technologies, sensing strategies, as well as analytical instrumentation and procedures. In the present study, we tested the applicability of a tapered optical fiber biosensor to detect amplified target DNA. Here the target DNA was amplified through the LAMP reaction. LAMP is a nucleic acid amplification technique that is completely isothermal within 1 hour. As such, LAMP is the best detection method for various pathogens over other DNA amplification techniques, such as polymerase chain reaction analysis,. The LAMP assay utilized in this study was developed in a previous study to target the Leptospira secY gene. The outcome suggests the applicability of the tapered optical fiber biosensor to specifically detect the amplified target DNA, thus indicating the potential for integrating both techniques into a more robust biosensor.
The development of tapered optical fiber DNA biosensors is still in its infancy, and as such, it is important to address the inconsistencies and the manual nature of the functionalization process. Digital microfluidics has been in the limelight in the fast-evolving field of operation platforms for molecular nucleic acid diagnostics. Reagent consumption is reduced, detection limits are improved, and a faster reaction time is achieved after incorporating a biosensor in digital microfluidics devices,. More importantly, it is possible to fully automate the process while ensuring controlled chemical and biological reactions, which eventually reduce the overall cost and cross-contamination. This improved device will have potential application as an alternative nucleic acid detection tool for leptospirosis, particularly in countries where the disease is endemic. The nucleic acid-based approach for a biosensor can help with earlier and more accurate interventions and thus will benefit patient management, prevent unnecessary drug use, and reduce overall healthcare costs. The versatility of the LAMP assay developed to detect Leptospira in human, animal, and environmental samples will lead to expanded applicability of the biosensor. Given that leptospirosis is an environmentally-associated disease, the application of the biosensor to environmental samples will help with the environmental surveillance of bacteria to monitor, predict, and control disease outbreaks. The tapered optical fiber in the biosensor was a scaffold for the technology to develop biosensors for other diseases where a simple and cost-effective method is needed.
In conclusion, a Leptospira DNA detection platform based on a tapered optical fiber was presented herein, as molecular-based detection of Leptospira remains an effective diagnostic approach. The biosensor allowed for selective detection of Leptospira spp. by targeting the Leptospira secY gene, regardless of its pathogenicity classification. The findings demonstrated the detection of amplified DNA using the biosensor and thus suggest its integration in future work. These findings will pave the way for the future development of the biosensor as a new leptospirosis diagnostic paradigm that can be used in field and clinical settings.
Conflict of interest statement
The author declare that they have no conflict of interest.
The authors would like to express their gratitude to Universiti Putra Malaysia for supporting the work. The authors are also thankful to Associate Professor Dr. Vasantha Kumari Neela and Noraini Philip for providing the pure bacteria cultures.
This research was funded by Universiti Putra Malaysia through the Geran Inisiatif Putra Siswazah (GP-IPS/2019/9678200).
JYL, MHY and HYC conceptualized and designed the study. JYL collected the data and performed analysis and interpretation of the data. MHY and HYC provided the software for analysis and validated the analysis. JYL wrote the original draft. JYL, MHY and HYC have critically reviewed, revised, and approved the final version of the manuscript. MHY and HYC supervised the project. HYC acquired the funding.
| References|| |
Lau CL, Smythe LD, Craig SB, Weinstein P. Climate change, flooding, urbanisation and leptospirosis: Fuelling the fire? Trans R Soc Trop Med Hyg
Levett PN. Leptospirosis. Clin Microbiol Rev
McBride AJA, Athanazio DA, Reis MG, Ko AI. Leptospirosis. Curr Opin Infect Dis
Karande S, Bhatt M, Kelkar A, Kulkarni M, De A, Varaiya A. An observational study to detect leptospirosis in Mumbai, India, 2000. Arch Dis Child 2003
LaRocque RC, Breiman RF, Ari MD, Morey RE, Janan FA, Hayes JM, et al. Leptospirosis during dengue outbreak, Bangladesh. Emerg Infect Dis
Suppiah J, Chan SY, Ng MW, Khaw YS, Ching SM, Mat-Nor LA, et al. Clinical predictors of dengue fever co-infected with leptospirosis among patients admitted for dengue fever-A pilot study. J Biomed Sci
Vinetz JM. Leptospirosis. Curr Opin Infect Dis
Musso D, La Scola B. Laboratory diagnosis of leptospirosis: A challenge. J Microbiol Immunol Infect
Waggoner JJ, Pinsky BA. Molecular diagnostics for human leptospirosis. Curr Opin Infect Dis
Berney H, West J, Haefele E, Alderman J, Lane W, Collins JK. A DNA diagnostic biosensor: Development, characterisation and performance. Sens Actuators B Chem
Bhalla N, Jolly P, Formisano N, Estrela P. Introduction to biosensors. Essays Biochem
Tian Y, Wang W, Wu N, Zou X, Wang X. Tapered optical fiber sensor for label-free detection of biomolecules. Sensors
Bosch ME, Sánchez AJR, Rojas FS, Ojeda CB. Recent development in optical fiber biosensors. Sensors
Kamil YM, Bakar MHA, Mustapa MA, Yaacob MH, Syahir A, Mahdi MA. Sensitive and specific protein sensing using single-mode tapered fiber immobilized with biorecognition molecules. IEEE Photon J
Leung A, Shankar PM, Mutharasan R. A review of fiber-optic biosensors. Sens Actuators B Chem
Yadav TK, Narayanaswamy R, Abu Bakar MH, Kamil YM, Mahdi MA. Single mode tapered fiber-optic interferometer based refractive index sensor and its application to protein sensing. Opt Express
Zainuddin NH, Chee HY, Ahmad MZ, Mahdi MA, Abu Bakar MH, Yaacob MH. Sensitive Leptospira
DNA detection using tapered optical fiber sensor. J Biophoton
Marazuela D, Moreno-Bondi MC. Fiber-optic biosensors-an overview. Anal Bioanal Chem
Huang Y, Tian Z, Sun LP, Sun D, Li J, Ran Y, et al. High-sensitivity DNA biosensor based on optical fiber taper interferometer coated with conjugated polymer tentacle. Opt Express
Wang YM, Pang XF, Zhang YY. Recent advances in fiber-optic DNA biosensors. J Biomed Sci Eng
Wang X, Son A. Effects of pretreatment on the denaturation and fragmentation of genomic DNA for DNA hybridization. Environm Sci Process Impacts
Philip N, Affendy NB, Masri SN, Yuhana MY, Than LTL, Sekawi Z, et al. Combined PCR and MAT improves the early diagnosis of the biphasic illness leptospirosis. PLoS One
Othman S, Lee PY, Lam JY, Philip N, Azhari NN, Affendy NB, et al. A versatile isothermal amplification assay for the detection of leptospires from various sample types. Peer J
Ozkumur E, Ahn S, Yalçin A, Lopez CA, Cevik E, Irani RJ, et al. Label-free microarray imaging for direct detection of DNA hybridization and single-nucleotide mismatches. Biosens Bioelectron
Zuerner RL, Hartskeerl RA, van de Kemp H, Bal AE. Characterization of the Leptospira interrogans
S10-spc-alpha operon. FEMS Microbiol Lett
Ahmed A, Engelberts MFM, Boer KR, Ahmed N, Hartskeerl RA. Development and validation of a real-time PCR for detection of pathogenic Leptospira
species in clinical materials. PLoS One
Waggoner JJ, Balassiano I, Abeynayake J, Sahoo MK, Mohamed-Hadley A, Liu Y, et al. Sensitive real-time PCR detection of pathogenic Leptospira
spp. and a comparison of nucleic acid amplification methods for the diagnosis of leptospirosis. PLoS One
Alizadeh S, Javadi A, Najafipour R, Farivar T. Simultaneous detection of pathogenic and saprophyte Leptospira
in human plasma by multiplex taqman real time PCR. Biotechnol Health Sci
Lam JY, Low GKK, Chee HY. Diagnostic accuracy of genetic markers and nucleic acid techniques for the detection of Leptospira
in clinical samples: A meta-analysis. PLoS Neglect Trop Dis
(2). doi: 10.1371/journal.pntd.0008074.
Leung A, Shankar PM, Mutharasan R. Label-free detection of DNA hybridization using gold-coated tapered fiber optic biosensors (TFOBS) in a flow cell at 1310 nm and 1550 nm. Sens Actuat B
Wang X, Cooper KL, Wang A, Xu J, Wang Z, Zhang Y, et al. Label-free DNA sequence detection using oligonucleotide functionalized optical fiber. Appl Phys Lett
Mustapha Kamil Y, Al-Rekabi SH, Yaacob MH, Syahir A, Chee HY, Mahdi MA, et al. Detection of dengue using PAMAM dendrimer integrated tapered optical fiber sensor. Sci Rep
Tabata M, Yao B, Seichi A, Suzuki K, Miyahara Y. Electrochemical biosensors combined with isothermal amplification for quantitative detection of nucleic acids. Method, Molecul Biol
Toubanaki DK, Athanasiou E, Karagouni E. Gold nanoparticle-based lateral flow biosensor for rapid visual detection of Leishmania-specific
DNA amplification products. J Microbiol Methods
Yan Y, Ding S, Zhao D, Yuan R, Zhang Y, Cheng W. Direct ultrasensitive electrochemical biosensing of pathogenic DNA using homogeneous target-initiated transcription amplification. Sci Rep
Vasdev K. DNA biosensors-A review. J Bioeng Biomed Sci
2017; doi: 10.4172/2155-9538.1000222.
Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, et al. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res
Wong YP, Othman S, Lau YL, Radu S, Chee HY. Loop-mediated isothermal amplification (LAMP): A versatile technique for detection of micro-organisms. J Appi Microbiol
Njiru ZK. Loop-mediated isothermal amplification technology: Towards point of care diagnostics. PLoS Negl Trop Dis
Coelho B, Veigas B, Fortunato E, Martins R, Águas H, Igreja R, et al. Digital microfluidics for nucleic acid amplification. Sensors
Norian H, Field RM, Kymissis I, Shepard KL. An integrated CMOS quantitative-polymerase-chain-reaction lab-on-chip for point-of-care diagnostics. Lab Chip
Kalsi S, Valiadi M, Tsaloglou MN, Parry-Jones L, Jacobs A, Watson R, et al. Rapid and sensitive detection of antibiotic resistance on a programmable digital microfluidic platform. Lab
Chang YH, Lee GB, Huang FC, Chen YY, Lin JL. Integrated polymerase chain reaction chips utilizing digital microfluidics. Biomed Microdev
The Publisher of the Journal remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]