A novel magnetic ligand-based assay for the electrochemical determination of BRD4.

The first magnetic ligand-based electrochemical assay aimed at the determination of BRD4 was developed and validated. BRD4 is an epigenetic regulator of great interest in oncology in relation to its overexpression observed in the pathogenesis of several cancer diseases. BRD4 also represents a major target for the development of innovative treatments aimed at protein inhibition or degradation. Despite the relevance of BRD4 both for diagnostics and therapeutic purposes, current methodologies for its determination are limited to commercial ELISA kits. We present a novel magnetic ligand-based assay for the electrochemical determination of BRD4. The developed assay is based on the use of a small synthetic fragment of the natural protein ligand for BRD4 as receptor, thus exploiting the intrinsic biological protein-protein recognition mechanism. In addition, the assay features the use of magnetic beads as immobilization platforms and peroxidase-conjugated monoclonal anti-BRD4 antibody for the generation of the electrochemical signal. The ligand-based assay shows outstanding performance in terms of rapidity, with results achievable in less than 20 min, no matrix effect when applied to human plasma or cell lysate samples, and excellent specificity. The proposed method exhibits a limit of detection of 2.66 nM and a response range tunable as a function of the amount of immobilized receptor. The developed ligand-based assay was successfully applied to the accurate determination of BRD4 in untreated cell lysates, as proven by the ELISA reference method. The good performance of the proposed bioassay for determination of BRD4 showed potential application of this strategy in convenient point-of-care testing.

Ultrasensitive PCR-Free detection of whole virus genome by electrochemiluminescence.

Detection of nucleic acids is crucial in many medical applications, and in particular for monitoring infectious diseases, as it has become perfectly clear after the pandemic infection of COVID-19. In this context, the development of innovative detection methods based on signal-amplification rather than analyte-amplification represents a significant breakthrough compared to existing PCR-based methodologies, allowing the development of new nucleic acid detection technologies suitable to be integrated in portable and low-cost sensor devices while keeping high sensitivities, thus enabling massive diagnostic screening. In this work, we present a novel molecular sensor for the ultrasensitive PCR-free detection of Hepatitis B Virus (HBV) based on electrochemiluminescence (ECL). Thanks to the combination of surface cooperative hybridization scheme with ECL detection strategy, our novel DNA sensor is able to detect HBV genome - both synthetic and extracted - with the unprecedented limit of detection (LoD) of 0.05 cps µL-1 for extracted sample, that is even lower than the typical LoD of PCR methodologies. The detection concept presented here for HBV detection is very versatile and can be extended to other pathogens, paving the way for future development of rapid molecular test for infectious diseases, both viral and bacterial, in Point-of-Care (PoC) format.

Application of chemometrics for detection and modeling of adulteration of fresh cow milk with reconstituted skim milk powder using voltammetric fingerpriting on a graphite/ SiO2 hybrid electrode.

In the present work, an analytical approach for the voltammetric detection and prediction of adulteration of fresh cow milk with reconstituted skim milk powder is developed. After precipitation of milk proteins upon addition of ethanol and centrifugation, the supernatant liquid of the samples was analyzed by cyclic voltammetry on a novel graphite/SiO2 hybrid working electrode (GSiHE) using LiClO4 as electrolyte. Under these conditions, fresh milk samples gave broadened peaks/plateaus in both forward and backward potential scanning, attributed mainly to oxidases. Such peaks were not evident in the case of reconstituted skim milk powder samples due to inactivation of enzymes and breakdown of certain antioxidants caused by heat and pressure-treatments. The differences between fresh and reconstituted skim milk powder samples in their voltammetric profile were exploited for the detection of fresh milk adulteration by submitting voltammetric data to chemometrics. As datapoints, the differences between forward and backward current values, recorded at the same potentials, were determined and submitted to multivariate analysis. Principal Component Analysis (PCA) provided a clear differentiation between fresh milk and reconstituted skim milk powder samples. Soft independent modeling of class analogy (SIMCA) was employed to model the class of fresh milks, using samples from 12 commercially available fresh milk brands. Prediction of fresh milk adulteration with reconstituted skim milk powders was achieved by means of Partial Least Squares (PLS) regression analysis. Detection limit of the technique was found to be below 6% (v/v) and the linearity of model in terms of observed/predicted values was confirmed up to 100% (v/v). Validation and applicability of both SIMCA and PLS models were confirmed using a suitable test set, consisting of commercial fresh milk and skim milk powder samples as well as synthetic adulterated fresh milk samples.