SIRT7 gene knockout using CRISPR/Cas9 system enhances melanin production in the melanoma cells.

Melanin is a prominent pigment of skin and hair, and its deficiency can cause various disorders such as hair graying and albinism. The improvement of melanin production at a genetic level could offer an effective and permanent solution. Recently, SIRT7 has evoked an interest in the study of hair follicle stem cells, but its role in melanin synthesis remains unclear. In the present study, we have first successfully developed SIRT7 gene KO melanoma cells using the CRISPR/Cas9 system. It was found that the SIRT7 gene KO enhanced melanin production in melanoma cells. To validate the role of SIRT7 in melanin production, RT-PCR, western blot, and immunofluorescence staining assays were performed. The expression levels of melanin-producing genes and proteins (MITF, TRP1, TRP-2, TYR, TH) were significantly increased in SIRT7 gene KO cells compared to normal cells. In addition, melanin production was increased in KO cells higher than in normal cells through the image analysis. All these results suggest that SIRT7 could play an essential role in regulating melanin production, providing an alternative drug target to treat pigmentary disorders.

Rapid and sensitive detection of melanin using glutathione conjugated gold nanocluster based fluorescence quenching assay.

In the present study, a rapid, facile, and highly sensitive assay based on glutathione conjugated gold nanocluster (GSH-AuNCs) is developed for the detection of melanin. The analysis of melanin which is linked to several diseases is crucial. The current methods for melanin estimation are complex and long, thus demands an alternative technology. In general, melanin exhibits photoactive properties, thus, it might have fluorescence quenching properties through the phenomenon of fluorescence resonance energy transfer. To verify our assumption, we have developed the fluorescence quenching assay based on gold nanocluster and melanin interaction. As a result, under the optimized condition, the developed quenching assay demonstrated the high selectivity and sensitivity toward melanin with a limit of detection and correlation coefficient of 0.060 µg/mL and 0.993, respectively. Moreover, the whole process represented the rapid assay time of 30 min to complete. To validate the performance of our assay on real samples, B16F1 cells lysate, and hair samples were tested that provided satisfactory results. Therefore, we believe that our assay due to good sensitivity and short assay time could be beneficial for the clinical diagnosis of melanin in the future study.

Detection of C-Reactive Protein Using Histag-HRP Functionalized Nanoconjugate with Signal Amplified Immunoassay.

Ultrasensitive detection of biomarkers is highly significant for disease prognosis and public health treatment. Despite wide acceptance in routine laboratory tests, the conventional enzyme-linked immunosorbent assay (ELISA) has been of limited use for early biomarker detection due to insufficient sensitivity and multiple long incubation time. Several nanoprobes have been introduced to circumvent the limitation, however, rapid, simple, and chemical-free nanoprobe synthesis and sensitive detection methods, particularly for ELISA, are still lacking. In this study, we have synthesized a gold nanoprobe, conjugated with multiple 6X-histidine (6X-his) peptide and nickel-horseradish peroxidase (Ni2+-HRP), for enhancing the colorimetric signal in ELISA. The developed nanoprobe has been tested for the detection of immunologically significant C-reactive protein (CRP) in ELISA format. The performance of designed probe is validated by testing standard and serum samples, and the detection limit of 32.0 pg/mL with R2 = 0.98 is confirmed. Furthermore, a comparative analysis of the developed nanoprobe was performed with ELISA developed on conventional guidelines, the proposed immunoassay showed an increase of 12-fold sensitivity for detecting CRP due to the high loading of 6Xhis peptide and binding of multiple Ni2+-HRP on a gold nanoparticle. Additionally, the proposed assay provides a simple, fast, and cost-efficient (not requiring multiple antibodies) detection of CRP with easy nanoprobe synthesis. Moreover, the developed Histag-HRP functionalized nanoconjugate immunoassay is flexible and can be applied to other biomarkers efficiently by using disease specific antibody.

Tannic acid-mediated rapid layer-by-layer deposited non-leaching silver nanoparticles hybridized cellulose membranes for point-of-use water disinfection.

Gravity driven water filtration is a commonly used process of removing microorganism from the contaminated water. However, the existing strategies involve prolonged synthesis and toxic reducing agent for immobilization of silver nanoparticles (AgNPs) on cellulose membranes that are not suitable for routine handling. We have developed a non-toxic and environmentally benign method using TA mediated silver salt layer-by-layer (LbL) in-situ reduction method. Our LbL method exhibited the properties of controlled size and uniform distribution of in-site AgNPs on the surface of the membranes. The LbL deposited AgNPs hybrid membranes displayed an excellent antibacterial activity which have been validated through an efficient bacterial filtration performance against the Escherichia coli (E. coli). The present method for developing hybrid membranes offered a simple, rapid, low-cost, sustainable, and large-scale fabrication for bacterial filtration which could be used for the point-of-use applications, particularly at resource-limited and remote areas.

Miniaturized Sample Preparation and Rapid Detection of Arsenite in Contaminated Soil Using a Smartphone.

Conventional methods for analyzing heavy metal contamination in soil and water generally require laboratory equipped instruments, complex procedures, skilled personnel and a significant amount of time. With the advancement in computing and multitasking performances, smartphone-based sensors potentially allow the transition of the laboratory-based analytical processes to field applicable, simple methods. In the present work, we demonstrate the novel miniaturized setup for simultaneous sample preparation and smartphone-based optical sensing of arsenic As(III) in the contaminated soil. Colorimetric detection protocol utilizing aptamers, gold nanoparticles and NaCl have been optimized and tested on the PDMS-chip to obtain the high sensitivity with the limit of detection of 0.71 ppm (in the sample) and a correlation coefficient of 0.98. The performance of the device is further demonstrated through the comparative analysis of arsenic-spiked soil samples with standard laboratory method, and a good agreement with a correlation coefficient of 0.9917 and the average difference of 0.37 ppm, are experimentally achieved. With the android application on the device to run the experiment, the whole process from sample preparation to detection is completed within 3 hours without the necessity of skilled personnel. The approximate cost of setup is estimated around 1 USD, weight 55 g. Therefore, the presented method offers the simple, rapid, portable and cost-effective means for onsite sensing of arsenic in soil. Combined with the geometric information inside the smartphones, the system will allow the monitoring of the contamination status of soils in a nation-wide manner.

A low cost design and fabrication method for developing a leak proof paper based microfluidic device with customized test zone.

This article describes a fabrication process for the generation of a leak proof paper based microfluidic device and a new design strategy for convenient incorporation of externally prepared test zones. Briefly, a negative photolithographic method was used to prepare the device with a partial photoresist layer on the rear of the device to block the leakage of sample. Microscopy and Field Emission Scanning Electron Microscopy data validated the formation of the photoresist layer. The partial layer of photoresist on the device channel limits sample volume to 7 ± 0.2 µl as compared to devices without the partial photoresist layer which requires a larger sample volume of 10 ± 0.1 µl. The design prototype with a customized external test zone exploits the channel protrusions on the UV exposed photoresist treated paper to bridge the externally applied test zone to the sample and absorbent zones. The partially laminated device with an external test zone has a comparatively low wicking speed of 1.8 ± 0.9 mm/min compared to the completely laminated device with an inbuilt test zone (3.3 ± 1.2 mm/min) which extends the reaction time between the analyte and reagents. The efficacy of the prepared device was studied with colorimetric assays for the non-specific detection of protein by tetrabromophenol blue, acid/base with phenolphthalein indicator, and specific detection of proteins using the HRP-DAB chemistry. The prepared device has the potential for leak proof detection of analyte, requires low sample volume, involves reduced cost of production (∼$0.03, excluding reagent and lamination cost), and enables the integration of customized test zones.