Electrochemical biosensor based on cellulose nanofibers/graphene oxide and acetylcholinesterase for the detection of chlorpyrifos pesticide in water and fruit juice.

In this work, cellulose nanofibers and graphene oxide are used to fabricate a simple and reliable electrochemical biosensor, based on acetylcholinesterase (AChE) for the detection of highly dangerous organophosphates (OPs), utilizing chlorpyrifos as a representative sample. AChE is an enzyme that is essential for neurotransmission and catalyzes the conversion of acetylcholine (ATCh) into thiocholine and acetic acid. The pesticide used in this work, chlorpyrifos, inhibits the catalytic activity of AChE on ATCh, and this inhibition can be measured using square wave voltammetry (SWV). Utilizing a process of surface modification, layers of cellulose nanofibers, graphene oxide, a chitosan-graphene oxide composite, and acetylcholinesterase (AChE/CS-GO/GO/CNFs) were immobilized on a screen-printed carbon electrode (SPCE). The modified SPCE working electrode was characterized using scanning electron microscopy and graphene oxide trapped in the cellulose nanofibers was found to increase the sensitivity of the biosensor. The modified biosensor demonstrated good performance for detection of chlorpyrifos over a linear range of 25-1000 nM under optimum conditions, and the limits of detection and quantification were 2.2 nM and 73 nM, respectively. Our sophisticated technique might offer a more precise, straightforward, quick, and environmentally friendly way to assess chlorpyrifos contamination in water and juice samples.

A single-round multiplex PCR assay for the identification of Anopheles minimus related species infected with Plasmodium falciparum and Plasmodium vivax.

This study aimed to develop a single-round multiplex PCR method for the identification of Anopheles minimus complex (An. minimus and Anopheles harrisoni) and Anopheles aconitus subgroup (An. aconitus and Anopheles varuna), and for the simultaneous detection of Plasmodium falciparum and Plasmodium vivax in these vectors. Five primers were created for a single-round multiplex PCR assay to identify four anopheline mosquitoes combined with three Plasmodium primers for the detection of P. falciparum and P. vivax in vectors. The four species of anopheline vectors and two Plasmodium species, P. falciparum and P. vivax, could be identified by the combination of eight primers in the single-round multiplex PCR assay. The amplified species-specific products were 380bp for An. minimus, 180bp for An. harrisoni, 150bp for An. aconitus, 310bp for An. varuna, 276bp for P. falciparum, and 300bp for P. vivax. The sensitivities were 0.5pg/µl (25sporozoites/µl) for P. falciparum DNA and between 0.5 and 5pg/µl (25-250sporozoites/µl) for P. vivax DNA. Furthermore, this developed method could be used to identify field caught An. minimus complex, An. aconitus subgroup from Thailand and Lao PDR. Also, it was successfully used to identify the species An. minimus, An. harrisoni, An. aconitus and An. varuna and to detect and identify P. falciparum and P. vivax in caught anopheline mosquitoes. The sensitivity of this method was high for simultaneous detection of P. falciparum and P. vivax in anopheline mosquitoes.

The development of DNA-based quartz crystal microbalance integrated with isothermal DNA amplification system for human papillomavirus type 58 detection.

To address the effect of dramatic change in temperature and viscosity during PCR process on quartz crystal microbalance (QCM) sensor and to increase the sensitivity, isothermal amplification was employed in the system. We combined loop-mediated isothermal amplification (LAMP) technique with QCM, called as LAMP-QCM, for detection of high-risk human papillomavirus viral DNA type 58 (HPV-58) which is commonly found in Asian women. The liquid-phase LAMP-QCM prototype comprised the frequency counter, a temperature control device and housing of the quartz crystal with polished gold electrodes on both sides. QCM detection signal was monitored in real-time based on an avidin-biotin binding between avidin coated QCM surface and specific biotinylated LAMP products. Analytical performance was evaluated for precision, sensitivity and specificity. A plasmid clone containing the HPV-58 sequence was diluted from 10(6) to 1 copy and used for detection limit. Cut-off value was estimated at 28.8 Hz from negative viral template. The system could detect 100 copies with Δf at 34.0±3.6 Hz compared to 1000 copies detected by conventional LAMP. No cross-reaction was observed with other HPV types. The HPV-58 detection was compared among LAMP-QCM, conventional LAMP and nested PCR in 50 cervical cancer tissues. The positive rate of LAMP-QCM was higher than that of conventional LAMP with 100% sensitivity and 90.5% specificity. The integrated LAMP-QCM system has improved the detection limit up to ten times compared to conventional LAMP with less-time consuming.