Reverting TP53 Mutation in Breast Cancer Cells: Prime Editing Workflow and Technical Considerations.

Breast cancer is the leading cause of cancer-related deaths in women. The aggressive breast cancer subtype is commonly linked to the genetic alterations in the TP53 tumor suppressor gene, predominantly the missense mutations. Robust experimental models are needed to gain better insights into these mutations' molecular properties and implications in tumorigenesis. The generation of such models harboring the alterations is feasible with the CRISPR-based gene editing technology. Moreover, the development of new CRISPR applications, particularly DNA base and prime editing, has considerably improved the precision and versatility of gene editing. Here, we employed the prime editing tool to revert a TP53 missense C > T mutation (L194F) in a T47D luminal A breast cancer cell line. In parallel, this prime editing tool was also utilized to introduce the L194F mutation in HEK293T cells. To assess the prime editing efficiency in both cell lines, we first performed Sanger sequencing in the prime-edited cells pool and single cell-derived clones. However, the Sanger sequencing approach did not detect any base substitution in these cell lines. Next, by employing the more sensitive amplicon target sequencing, we managed to identify the expected substitution in these T47D and HEK293T cells, albeit the editing efficiency was low. In light of these findings, we discussed the technical aspects and provided suggestions for improve the prime editing workflow and efficiency for future experiments.

Bio-nanogate manipulation on electrode surface as an electrochemical immunosensing strategy for detecting anti-hepatitis B surface antigen.

The diagnosis of hepatitis B virus (HBV) and monitoring of the vaccination efficiency against HBV require real-time analysis. The presence of antibody against hepatitis B virus surface antigen (anti-HBsAg) as a result of HBV infection and/or immunization may indicate individual immune status towards HBV. This study investigated the ability of a bio-nanogate-based displacement immunosensing strategy in detecting anti-HBsAg antibody, via nonspecific-binding between polyamidoamine dendrimers encapsulated gold nanoparticles (PAMAM-Au) and the 'antigenic determinant' region (aD) of HBsAg. For this purpose, maltose binding protein harbouring the aD region (MBP-aD) was synthesized as a bioreceptor and immobilized on the screen-printed carbon electrode (SPCE). Following that, PAMAM-Au was deposited on MBP-aD, forming the 'gate' and was used as a monitoring agent. Under optimal conditions, the high specificity of anti-HBsAg antibody towards MBP-aD displaced PAMAM-Au causing the decrement of anodic peak in differential pulse voltammetry (DPV) analysis. The signal changes were proportionally related to the concentration of anti-HBsAg antibody, in a range of 1 - 1000 mIU/mL with a limit of detection (LOD) of 2.5 mIU/mL. The results also showed high specificity and selectivity of the immunosensor platform in detecting anti-HBsAg antibody both in spiked buffer and human serum samples.

Current Development in Decolorization of Synthetic Dyes by Immobilized Laccases.

The world today is in a quest for new means of environmental remediation as the methods currently used are not sufficient to halt the damage. Mostly, a global direction is headed toward a shift from traditional chemical-based methods to a more ecofriendly alternative. In this context, biocatalysis is seen as a cost-effective, energy saving, and clean alternative. It is meant to catalyze degradation of recalcitrant chemicals in an easy, rapid, green, and sustainable manner. One already established application of biocatalysis is the removal of dyes from natural water bodies using enzymes, notably oxidoreductases like laccases, due to their wide range of substrate specificity. In order to boost their catalytic activity, various methods of enhancements have been pursued including immobilization of the enzyme on different support materials. Aside from increased catalysis, immobilized laccases have the advantages of higher stability, better durability against harsh environment conditions, longer half-lives, resistance against protease enzymes, and the ability to be recovered for reuse. This review briefly outlines the current methods used for detoxification and decolorization of dye effluents stressing on the importance of laccases as a revolutionary biocatalytic solution to this environmental problem. This work highlights the significance of laccase immobilization and also points out some of the challenges and opportunities of this technology.

Interaction study of peptide-PAMAM as potential bio-nanogate for detecting anti-hepatitis B surface antigen.

Bio-nanogate involves synthesized or natural molecules as a 'gate' towards bioreceptors and responds upon the presence of targeted analytes in nanoscale dimension. Development of bio-nanogate improves analyte selectivity and signal response across various types of biosensors. The versatility of PAMAM dendrimers to form conjugates with guest molecules, such as proteins can be utilized in forming a bio-nanogate. PAMAM interaction with peptide bioreceptor for antibody detection is of interest in this study. This study investigated the interaction of synthesized immunogenic 'a' determinant (aD) region of hepatitis B virus surface antigen (HBsAg) with PAMAM G4 and anti-HBsAg antibody, as a potential bio-nanogate for anti-HBsAg detection. The aD peptide fused with maltose binding protein (MBP), was confirmed with Western blotting. Nano-Differential Scanning Fluorimetry (nano-DSF) study revealed that the interaction of MBP-aD with anti-HBsAg indicated a higher thermal stability as compared to its interaction with PAMAM G4. Electrochemical impedance spectroscopy showed that a higher binding constant of MBP-aD interaction with anti-HBsAg (0.92 µM-1) was observed at maximum saturation, as compared with PAMAM G4 (0.07 µM-1). Thermodynamic parameters demonstrated that MBP-aD interacted with anti-HBsAg and PAMAM G4, through van der Waals and hydrogen bonding. These analyses suggest that the weak interaction of MBP-aD and PAMAM G4 may form a potential bio-nanogate. It is hypothesized that the presence of anti-HBsAg has a higher affinity towards MBP-aD which may displace PAMAM G4 in the anti-HBsAg detection system. This interaction study is crucial as an initial platform of using peptide-PAMAM as a bio-nanogate in an antibody detection system.

Gold nanoparticle-decorated reduced-graphene oxide targeting anti hepatitis B virus core antigen.

Hepatitis B virus core antigen (HBcAg) is the major structural protein of hepatitis B virus (HBV). The presence of anti-HBcAg antibody in a blood serum indicates that a person has been exposed to HBV. This study demonstrated that the immobilization of HBcAg onto the gold nanoparticles-decorated reduced graphene oxide (rGO-en-AuNPs) nanocomposite could be used as an antigen-functionalized surface to sense the presence of anti-HBcAg. The modified rGO-en-AuNPs/HBcAg was then allowed to undergo impedimetric detection of anti-HBcAg with anti-estradiol antibody and bovine serum albumin as the interferences. Upon successful detection of anti-HBcAg in spiked buffer samples, impedimetric detection of the antibody was then further carried out in spiked human serum samples. The electrochemical response showed a linear relationship between electron transfer resistance and the concentration of anti-HBcAg ranging from 3.91ngmL-1 to 125.00ngmL-1 with lowest limit of detection (LOD) of 3.80ngmL-1 at 3σm-1. This established method exhibits potential as a fast and convenient way to detect anti-HBcAg.

A review on visible-light induced photoelectrochemical sensors based on CdS nanoparticles.

Discovering the distinctive photophysical properties of semiconductor nanoparticles (NPs) has made these a popular subject in recent advances in nanotechnology-related analytical methods. Semiconductor NPs are well-known materials that have been widely used in photovoltaic devices such as optical sensors and bioimaging, and dye-sensitized solar cells (DSSCs), as well as for light-emitting diodes (LEDs). The use of a narrow-bandgap semiconductor such as CdS NPs in the photoelectrochemical (PEC) detection of chemicals and biological molecules plays a key role as a photosensitizer and promotes some specific advantages in light-harvesting media. Their size-controlled optical and electrical properties make NPs fascinating and promising materials for a variety of nanoscale photovoltaic devices. Moreover, charge injection from the narrow bandgap to the adjacent material leads to efficient charge separation and prolongs the electron lifetime by the elimination of the charge carrier recombination probability. In this regard, a single photon enables the production of multiple photogenerated charge carriers in CdS NPs, which subsequently boosts the effectiveness of the photovoltaic devices. In particular, the present review article highlights the recent emerging PEC detection methods based on CdS NPs, specifically related to the direct and indirect interactions of NPs with target analytes. The current opportunities and challenges in achieving real-world applications of CdS-based PEC sensing are also presented.

Electrochemical detection of uric acid via uricase-immobilized graphene oxide.

Measurement of the uric acid level in the body can be improved by biosensing with respect to the accuracy, sensitivity and time consumption. This study has reported the immobilization of uricase onto graphene oxide (GO) and its function for electrochemical detection of uric acid. Through chemical modification of GO using 1-ethyl-3-(dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysulfosuccinimide (NHS) as cross-linking reagents, the enzyme activity of the immobilized uricase was much comparable to the free enzyme with 88% of the activity retained. The modified GO-uricase (GOU) was then subjected to electrocatalytic detection of uric acid (UA) via cyclic voltammetry (CV). For that reason, a glassy carbon electrode (GCE) was modified by adhering the GO along with the immobilized uricase to facilitate the redox reaction between the enzyme and the substrate. The modified GOU/GCE outperformed a bare electrode through the electrocatalytic activity with an amplified electrical signal for the detection of UA. The electrocatalytic response showed a linear dependence on the UA concentration ranging from 0.02 to 0.49 mM with a detection limit of 3.45 µM at 3σ/m. The resulting biosensor also exhibited a high selectivity towards UA in the presence of other interference as well as good reproducibility.

Enhanced biocatalytic esterification with lipase-immobilized chitosan/graphene oxide beads.

In this work, lipase from Candida rugosa was immobilized onto chitosan/graphene oxide beads. This was to provide an enzyme-immobilizing carrier with excellent enzyme immobilization activity for an enzyme group requiring hydrophilicity on the immobilizing carrier. In addition, this work involved a process for the preparation of an enzymatically active product insoluble in a reaction medium consisting of lauric acid and oleyl alcohol as reactants and hexane as a solvent. This product enabled the stability of the enzyme under the working conditions and allowed the enzyme to be readily isolated from the support. In particular, this meant that an enzymatic reaction could be stopped by the simple mechanical separation of the "insoluble" enzyme from the reaction medium. Chitosan was incorporated with graphene oxide because the latter was able to enhance the physical strength of the chitosan beads by its superior mechanical integrity and low thermal conductivity. The X-ray diffraction pattern showed that the graphene oxide was successfully embedded within the structure of the chitosan. Further, the lipase incorporation on the beads was confirmed by a thermo-gravimetric analysis. The lipase immobilization on the beads involved the functionalization with coupling agents, N-hydroxysulfosuccinimide sodium (NHS) and 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC), and it possessed a high enzyme activity of 64 U. The overall esterification conversion of the prepared product was 78% at 60 °C, and it attained conversions of 98% and 88% with commercially available lipozyme and novozyme, respectively, under similar experimental conditions.

MALDI-target integrated platform for affinity-captured protein digestion.

To address immunocapture of proteins in large cohorts of clinical samples high throughput sample processing is required. Here a method using the proteomic sample platform, ISET (integrated selective enrichment target) that integrates highly specific immunoaffinity capture of protein biomarker, digestion and sample cleanup with a direct interface to mass spectrometry is presented. The robustness of the on-ISET protein digestion protocol was validated by MALDI MS analysis of model proteins, ranging from 40 fmol to 1 pmol per nanovial. On-ISET digestion and MALDI MS/MS analysis of immunoaffinity captured disease-associated biomarker PSA (prostate specific antigen) from human seminal plasma are presented.

Noncovalent antibody immobilization on porous silicon combined with miniaturized solid-phase extraction (SPE) for array based immunoMALDI assays.

This paper presents a new strategy to combine the power of antibody based capturing of target species in complex samples with the benefits of microfluidic reverse phase sample preparation on an integrated sample enrichment target (RP-ISET) and the analysis speed of MALDI MS. The immunoaffinity step is performed on an in-house developed 3D-structured high surface area porous silicon (PSi) matrix, which allows efficient antibody immobilization by surface adsorption without any coupling agents in 30-60 min. The hydrophilic nature of the porous silicon surface at the molecular level displays a low adsorption of background peptides when exposed to complex digests or plasma samples, improving the conditions for the antigen specific extraction and subsequent readout. At the same time, the hydrophobic behavior, due to the nanostructured surface, of the PSi material facilitates liquid confinement during the assay. Using a footprint conforming to the standard for 384 well microplates, direct adaption of the protocol into standard sample handling robots is possible. The performance of the proposed immunoaffinity PSi-ISET immunoMALDI (iMALDI) assay was evaluated by specific detection of angiotensin I at a 10 femtomol level in diluted plasma samples (10 µL, 1 nM).

Acoustic whole blood plasmapheresis chip for prostate specific antigen microarray diagnostics.

The generation of high quality plasma from whole blood is of major interest for many biomedical analyses and clinical diagnostic methods. However, it has proven to be a major challenge to make use of microfluidic separation devices to process fluids with high cell content, such as whole blood. Here, we report on an acoustophoresis based separation chip that prepares diagnostic plasma from whole blood linked to a clinical application. This acoustic separator has the capacity to sequentially remove enriched blood cells in multiple steps to yield high quality plasma of low cellular content. The generated plasma fulfills the standard requirements (<6.0 x 10(9) erythrocytes/L) recommended by the Council of Europe. Further, we successfully linked the plasmapheresis microchip to our previously developed porous silicon sandwich antibody microarray chip for prostate specific antigen (PSA) detection. PSA was detected by good linearity (R(2) > 0.99) in the generated plasma via fluorescence readout without any signal amplification at clinically relevant levels (0.19-21.8 ng/mL).