Laboratory diagnosis and potential application of nucleic acid biosensor approach for early detection of dengue virus infections

Dengue fever is caused by the dengue virus, the genus of Flaviviridae virus family. Until now, there is no specific medication to kill the dengue virus and patients just solely depend on the treatment of the dengue infection symptoms. Thus, a highly sensitive and rapid diagnostic tool for early diagnosis of dengue virus is very desirable, especially in resource limited-condition. We briefly review pro and cons of existing diagnostic methods for the detection of dengue virus (virus isolation, PCR, NS1Ag, Serology). We also highlight the recent advances of the biosensor technology in the dengue diagnostic dengue as a promising point-of-care diagnostic in the future. The DNA based biosensor technology combined miniaturized sample preparation offers a good opportunity for the commercialized point of care testing for dengue diagnosis in the future

The development of silicon nanowire as sensing material and its applications

The application of silicon nanowire (SiNW) as a sensing nanomaterial for detection of biological and chemical species has gained attention due to its unique properties. In this review, a short description is also demonstrated on the synthesis techniques of SiNWs and recent progress on sensor development based on electrochemical methods, fluorescence field-effect transistors (FET), and surface-enhanced Raman scattering (SERS) spectroscopy. We also discussed the challenges of SiNW-based sensors in the future

Surface modifications to boost sensitivities of electrochemical biosensors using gold nanoparticles/silicon nanowires and response surface methodology approach

This work describes fabrication of a DNA electrochemical sensor utilized of gold nanoparticles/silicon nanowires/indium tin oxide (AuNPs/SiNWs/ITO) as a modified substrate for detection of dengue virus DNA oligomers using methylene blue (MB) as a redox indicator. The response surface methodology (RSM) was applied as one of the advanced optimization methods for fabrication of SiNWs/AuNPs/ITO electrode and immobilization of DNA probes to enhance the sensitivity of DNA detection. Several factors were successfully optimized using RSM, including volume of SiNWs, concentration of dithiopropionic acid (DTPA), volume of AuNPs, DNA probe concentration, and DNA probe immobilization time. RSM approach shows that AuNPs and DNA probe concentration were the prominent factors affecting on the MB current signal and immobilization of DNA probe on AuNPs/SiNWs surface. This new developed sensor was able to discriminate complementary target sequences, noncomplementary and single-base mismatch sequences, for DNA dengue virus detection

Development of DNA electrochemical sensor based on silicon nanowires/gold nanoparticles-modifed electrode for early detection of dengue virus

A new DNA electrochemical sensor based on silicon nanowires (SiNWs) and gold nanoparticles (AuNPs) modified electrode was developed for dengue virus detection. In this study, two different fabricated electrodes; SiNWs/AuNPs-modified Indium tin oxide (ITO) and SiNWs/AuNPs-modified screen printed gold electrode (SPGE) have been fabricated. Field Emission Scanning Electron Microscope (FE-SEM) and Energy Dispersive X-ray Spectroscopy (EDX) analysis confirmed that the SiNWs/AuNPsnanocomposite was deposited and uniformly distribution on the surface of ITO and SPGE. Based on cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) characterization, the fabricated SiNWs/AuNPs-ITO and SiNWs/AuNPs-SPGE have shown a good electrical conductivity compared to unmodified electrode. SiNWs/AuNPs nanocomposite was further explored as DNA matrix for DNA probe immobilization where dengue virus oligonucleotide was used as bio-sensing model to evaluate the performances of DNA electrochemical sensor. Electrochemical detection of hybridization events between immobilized DNA probe and complementary sequences of dengue virus were monitored by Different pulse voltammetry (DPV) technique using methylene blue (MB) as a redox indicator. The decrease of MB peak current was obtained after hybridization detection by both fabricated electrodes. The optimal performance of SiNWs/AuNPs-ITO and SiNWs/AuNPs-SPGE for electrochemical detection of dengue virus were obtained using response surface methodology (RSM): SiNWs volume (10.8 μL and 6 μL), dithiopropionic acid (DTPA) (0.52 mM and 0.45 μL), AuNPs volume (83 μL and 29 μL), DNA probe concentration (5.8 μM and 5 μM), immobilization time (14 hours and 10 hours), pH buffer (7.5 and 7.8), NaCl concentration (1.45 M and 0.80 M), hybridization temperature (45 °C and 42 °C) and incubation time (12 min and 10 min), respectively. Under optimized condition, developed DNA sensor showed a higher sensitivity of oligonucleotide detection as compared to the non-optimized condition. It was shown that the developed DNA sensors; SiNWs/AuNPs-ITO and SiNWs/AuNPs- SPGE were able to detect complementary oligonucleotide dengue virus as low as 0.0891 ng/μL (10 pM) and 0.0000891 ng/μL (10 fM), respectively. The stability studies also have shown that fabricated ssDNA/AuNPs/SiNWs-ITO and the ssDNA/AuNPs/SiNWs-SPGE could be stored at 4 °C for 10 weeks and 7 weeks, respectively. It was found that the MB current signal of both developed DNA sensors have increased after the hybridization of immobilized DNA probe with genomic dengue virus from cell culture samples. However, this finding was unclear to justify the ability of both developed DNA sensor for direct detection of genomic dengue virus because of MB binding interaction issue and high non-specific hybridization for long genomic sequences. Hence, the preparation of specific and amplified target genomic dengue virus using reverse-transcribe-polymerase chain reaction (RT-PCR) were investigated. The parameters of annealing temperature, sonication time and reverseforward (R/F) primer ratio using RT-PCR methods have been studied. Both developed DNA sensors are capable to discriminate the MB signal of blank electrode, negative serum samples, dengue 1 and 2 –spiked serum, cell culture and negative control. The LOD obtained for RT-PCR products value were 5.6 ng/μL and 2.8 ng/μL for SiNWs/AuNPs-ITO and SiNWs/AuNPs-SPGE, respectively. Furthermore, the developed DNA sensors; SiNWs/AuNPs-ITO and SiNWs/AuNPs-SPGE showed good reproducibility for nine measurements where the RSD value of 9.34 % and 8.23 % were obtained, respectively

The strategies of DNA immobilization and hybridization detection mechanism in the construction of electrochemical DNA sensor: A review

In recent years, electrochemical deoxyribonucleic acid (DNA) sensor has recently emerged as promising alternative clinical diagnostic devices especially for infectious disease by exploiting DNA recognition events and converting them into an electrochemical signal. This is because the existing DNA diagnostic method possesses certain drawbacks such as time-consuming, expensive, laborious, low selectivity and sensitivity. DNA immobilization strategies and mechanism of electrochemical detection are two the most important aspects that should be considered before developing highly selective and sensitive electrochemical DNA sensor. Here, we focus on some recent strategies for DNA probes immobilization on the surface of electrochemical transducer such as adsorption, covalent bonding and Avidin/Streptavidin-Biotin interaction on the electrode surface for specific interaction with its complementary DNA target. A numerous approach for DNA hybridization detection based electrochemical technique that frequently used including direct DNA electrochemical detection and label based electrochemical (redox-active indicator, enzyme label and nanoparticles were also discussed in aiming to provide general guide for the design of electrochemical DNA sensor. We also discussed the challenges and suggestions to improve the application of electrochemical DNA sensor at point-care setting. Keywords: Electrochemical DNA sensor, DNA immobilization, DNA hybridization, Electrochemical mechanis

Strategies for the preparation of non-amplified and amplified genomic dengue gene samples for electrochemical DNA biosensing applications

The application of electrochemical DNA biosensors in real genomic sample detection is challenging due to the existence of complex structures and low genomic concentrations, resulting in inconsistent and low current signals. This work highlights strategies for the treatment of non-amplified and amplified genomic dengue virus gene samples based on real samples before they can be used directly in our DNA electrochemical sensing system, using methylene blue (MB) as a redox indicator. The main steps in this study for preparing non-amplified cDNA were cDNA conversion, heat denaturation, and sonication. To prepare amplified cDNA dengue virus genomic samples using an RT-PCR approach, we optimized a few parameters, such as the annealing temperature, sonication time, and reverse to forward (R/F) primer concentration ratio. We discovered that the generated methylene blue (MB) signals during the electrochemical sensing of non-amplified and amplified samples differ due to the different MB binding affinities based on the sequence length and base composition. The findings show that our developed electrochemical DNA biosensor successfully discriminates MB current signals in the presence and absence of the target genomic dengue virus, indicating that both samples were successfully treated. This work also provides interesting information about the critical factors in the preparation of genomic gene samples for developing miniaturized PCR-based electrochemical sensing applications in the future. We also discuss the limitations and provide suggestions related to using redox-indicator-based electrochemical biosensors to detect real genomic nucleic acid genes

The utilization of SiNWs/AuNPs-modified indium tin oxide (ITO) in fabrication of electrochemical DNA sensor

This work describes the incorporation of SiNWs/AuNPs composite as a sensing material for DNA detection on indium tin-oxide (ITO) coated glass slide. The morphology of SiNWs/AuNPs composite as the modifier layer on ITO was studied by scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDX). The morphological studies clearly showed that SiNWs were successfully decorated with 20 nm-AuNPs using self-assembly monolayer (SAM) technique. The effective surface area for SiNWs/AuNPs-modified ITO enhanced about 10 times compared with bare ITO electrode. SiNWs/AuNPs nanocomposite was further explored as a matrix for DNA probe immobilization in detection of dengue virus as a bio-sensing model to evaluate its performance in electrochemical sensors. The hybridization of complementary DNA was monitored by differential pulse voltammetry (DPV) using methylene blue (MB) as the redox indicator. The fabricated biosensor was able to discriminate significantly complementary, non-complementary and single-base mismatch oligonucleotides. The electrochemical biosensor was sensitive to target DNA related to dengue virus in the range of 9.0–178.0 ng/ml with detection limit of 3.5 ng/ml. In addition, SiNWs/AuNPs-modified ITO, regenerated up to 8 times and its stability was up to 10 weeks at 4 °C in silica gel

Strategies in the optimization of DNA hybridization conditions and its role in electrochemical detection of dengue virus (DENV) using response surface methodology (RSM)

In recent years, limited research has been conducted on enhancing DNA hybridization-based biosensor approaches using statistical models. This study explores the application of response surface methodology (RSM) to improve the performance of a DNA hybridization biosensor for dengue virus (DENV) detection. The biosensor is based on silicon nanowires decorated with gold nanoparticles (SiNWs/AuNPs) and utilizes methylene blue as a redox indicator. The DNA hybridization process between the immobilized DNA probe and the target DENV gene was monitored using differential pulse voltammetry (DPV) based on the reduction of methylene blue. Fourier-transform infrared spectroscopy (FTIR) and electrochemical impedance spectroscopy (EIS) were employed to confirm successful DNA hybridization events on the modified screen-printed gold electrode (SPGE) surface. Several parameters, including pH buffer, NaCl concentration, temperature, and hybridization time, were simultaneously optimized, with NaCl concentration having the most significant impact on DNA hybridization events. This study enhances the understanding of the role of each parameter in influencing DNA hybridization detection in electrochemical biosensors. The optimized biosensor demonstrated the ability to detect complementary oligonucleotide and amplified DENV gene concentrations as low as 0.0891 ng mL−1 (10 pM) and 2.8 ng mL−1 , respectively. The developed biosensor shows promise for rapid clinical diagnosis of dengue virus infection

Response surface methodology for optimization of nitrocellulose preparation from nata de coco bacterial cellulose for propellant formulation

Nitrocellulose (NC) has garnered significant interest among researchers due to its versatile applications, contingent upon the degree of nitration that modifies the cellulose structure. For instance, NC with a high nitrogen content, exceeding 12.5%, finds utility as a key ingredient in propellant formulations, while variants with lower nitrogen content prove suitable for a range of other applications, including the formulation of printing inks, varnishes, and coatings. This communication aims to present the outcomes of our efforts to optimize the nitration reaction of bacterial cellulose to produce high-nitrogen-content NC, employing the response surface methodology (RSM). Our investigation delves into the influence of the mole ratio of sulfuric and nitric acids, reaction temperature, and nitration duration on the nitrogen content of the resultant products. Utilizing a central composite design (CCD), we identified the optimal conditions for NC synthesis. Analysis of variance (ANOVA) underscored the substantial impact of these reaction conditions on the percentage of nitrogen content (%N) yield. By implementing the predicted optimal conditions—namely, a H2SO4:HNO3 mole ratio of 3:1, a reaction temperature of 35 °C, and a reaction period of 22 min—we successfully produced NC with a nitrogen content of 12.64%. Characterization of these products encompassed elemental analysis, Fourier transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD), thermal gravimetric analysis (TGA), and field emission scanning electron microscopy (FESEM)

An electrochemical sensor based on gold nanoparticles-functionalized reduced graphene oxide screen printed electrode for the detection of pyocyanin biomarker in Pseudomonas aeruginosa infection

Multidrug resistant Pseudomonas aeruginosa (P. aeruginosa) is known to be a problematic bacterium for being a major cause of opportunistic and nosocomial infections. In this study, reduced graphene oxide decorated with gold nanoparticles (AuNPs/rGO) was utilized as a new sensing material for a fast and direct electrochemical detection of pyocyanin as a biomarker of P. aeruginosa infections. Under optimal condition, the developed electrochemical pyocyanin sensor exhibited a good linear range for the determination of pyocyanin in phosphate-buffered saline (PBS), human saliva and urine at a clinically relevant concentration range of 1–100 μM, achieving a detection limit of 0.27 μM, 1.34 μM, and 2.3 μM, respectively. Our developed sensor demonstrated good selectivity towards pyocyanin in the presence of interfering molecule such as ascorbic acid, uric acid, NADH, glucose, and acetylsalicylic acid, which are commonly found in human fluids. Furthermore, the developed sensor was able to discriminate the signal with and without the presence of pyocyanin directly in P. aeruginosa culture. This proposed technique demonstrates its potential application in monitoring the presence of P. aeruginosa infection in patients