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- Employing bacteria machinery for antibiotic detection: Using DNA gyrase for ciprofloxacin detectionPublication . Cardoso, Ana Rita; Carneiro, Liliana P.T.; Cabral-Miranda, Gustavo; Bachmann, Martin F.; Sales, Maria Goreti FerreiraThis work describes a new successful approach for designing biosensors that detect antibiotics. It makes use of a biomimetic strategy, by employing the biochemical target of a given antibiotic as its biorecognition element. This principle was tested herein for quinolones, which target DNA gyrase in bacteria. Ciprofloxacin (CIPRO) was tested as a representative antibiotic from the quinolone group; the sensitivity of biosensor to this group was confirmed by checking the response to another quinolone antibiotic (norfloxacin, NOR) and to a non-quinolone antibiotic (ampicillin, AMP). The biorecognition element used was DNA gyrase attached by ionic interactions to a carbon support, on a working electrode on common screen-printed electrodes (SPEs). The response against antibiotics was tested for increasing concentrations of CIPRO, NOR or AMP, and following the subsequent electrical changes by electrochemical impedance spectroscopy. The DNAgyrase biosensor showed sensitive responses for CIPRO and NOR, for concentrations down to 3.02 nM and 30.2 nM, respectively, with a very wide response range for CRIPRO, up to 30.2 µM. Its response was also confirmed selective for quinolones, when compared to its response against AMP. Further comparison to an immunosensor of similar design (adding antibodies instead of DNA gyrase) was made, revealing favourable features for the new biomimetic biosensor with 1.52 nM of limit of detection (LOD). Overall, the new approach presented herein is simple and effective for antibiotic detection, displaying a selective response against a given antibiotic group. The use of bacterial machinery as biorecognition element in biosensors may also provide a valuable tool to study the mechanism of action in bacterial cells of new drugs. This is especially important in the development of new drugs to fight bacterial resistance.
- Molecular Imprinting on Nanozymes for Sensing ApplicationsPublication . Cardoso, Ana Rita; Frasco, Manuela F.; Serrano, Verónica; Fortunato, Elvira; Sales, Maria Goreti FerreiraAs part of the biomimetic enzyme field, nanomaterial-based artificial enzymes, or nanozymes, have been recognized as highly stable and low-cost alternatives to their natural counterparts. The discovery of enzyme-like activities in nanomaterials triggered a broad range of designs with various composition, size, and shape. An overview of the properties of nanozymes is given, including some examples of enzyme mimics for multiple biosensing approaches. The limitations of nanozymes regarding lack of selectivity and low catalytic efficiency may be surpassed by their easy surface modification, and it is possible to tune specific properties. From this perspective, molecularly imprinted polymers have been successfully combined with nanozymes as biomimetic receptors conferring selectivity and improving catalytic performance. Compelling works on constructing imprinted polymer layers on nanozymes to achieve enhanced catalytic efficiency and selective recognition, requisites for broad implementation in biosensing devices, are reviewed. Multimodal biomimetic enzyme-like biosensing platforms can offer additional advantages concerning responsiveness to different microenvironments and external stimuli. Ultimately, progress in biomimetic imprinted nanozymes may open new horizons in a wide range of biosensing applications.
- In-situ production of Histamine-imprinted polymeric materials for electrochemical monitoring of fishPublication . Serrano, Verónica; Cardoso, Ana Rita; Diniz, Mário; Sales, Maria Goreti FerreiraHistamine (HIS) is a major public health problem due to its toxic properties. High levels can cause a chronic toxicity as poisoning and can be used as a signal of food hygiene. Thus, a new electrochemical sensor for HIS detection in fish is presented herein, prepared by tailoring a molecularly imprinted polymer (MIP) sensing material on a gold screen-printed electrode (Au-SPEs), in which the polymeric film was generated in-situ. This film was obtained by electropolymerizing aniline under conditions that preserved the chemical structure of HIS. Raman spectroscopy followed the chemical changes occurring at each stage of the electrode modification. The device performance was assessed by evaluating the changes in electron transfer properties of a standard redox probe [Fe(CN)6]4−/[Fe(CN)6]3−, by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). EIS was also used to calibrate the sensor, being the standard solutions prepared under different background media (electrolyte or a blank sample of fish extract). The device displayed a linear response from 500 nM to 1 mM, with a limit of detection of 210 nM, and a selective behaviour against tyramine, another amine related to fish degradation. The sensing system was further employed to monitor the HIS content in samples in different time points of storage at ambient temperature. The obtained results were in agreement with the ELISA method, while offering more reproducible data. In general, the optimized sensor allowed reproducible and accurate analysis of fish samples subject to degradation and was completely assembled in-situ, in a very simple and straightforward approach. The device is low cost and suitable for further adaptation to on-site analysis, as required in food control.
- Novel and simple electrochemical biosensor monitoring attomolar levels of miRNA-155 in breast cancerPublication . Cardoso, Ana Rita; Moreira, Felismina; Fernandes, Rúben; Sales, GoretiThis work, describes for the first time, a simple biosensing design to yield an ultrasensitive electro-chemical biosensor for a cancer biomarker detection, miRNA-155, with linear response down to the attomolar range. MiRNA-155 was selected for being overexpressed in breast cancer. The biosensor was assembled in two stages: (1) the immobilization of the anti-miRNA-155 that was thiol modified on an Au-screen printed electrode (Au-SPE), followed by (2) blocking the areas of non-specific binding with mercaptosuccinic acid. Atomic force microscopy (AFM) and electrochemical tech-niques including cyclic voltammetry (CV), impedance spectroscopy (EIS) and square wave voltammetry (SWV) confirmed the surface modification of these devices and their ability to hybridize successfully and stably with miRNA-155. The final biosensor provided a sensitive detection of miRNA-155 from 10 aM to 1.0 nM with a low detection limit (LOD) of 5.7 aM in real human serum samples. Good results were obtained in terms of selectivity towards breast cancer antigen CA-15.3 and bovine serum albumin (BSA). Raw fluid extracts from cell-lines of melanoma did not affect the biosensor response (no significant change of the blank), while raw extracts from breast cancer yielded a positive signal against miRNA-155. This simple and sensitive strategy is a promising alternative for simultaneous quantitative analysis of multiple miRNA in physiological fluids for biomedical research and point-of-care (POC) diagnosis.