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  • Open access
  • 13 Reads
Staphylococcus aureus detection in milk using a Quartz Crystal Microbalance aptasensor with an antifouling probe linker.

Contamination of food through the presence of pathogens can pose a serious risk to health. For safe food consumption, monitoring for the presence of pathogens is critical to identify and regulate microbiological contamination. Milk is no exception and requires rapid analysis of samples to identify unsafe bacteria levels. In this work, an aptasensor based on a quartz crystal microbalance with dissipation monitoring was developed to detect and quantify Staphylococcus aureus directly in whole UHT cow’s milk. The aptasensor demonstrated high sensitivity and was able to detect S. aureus in milk with a 33 CFU/mL limit of detection. Analysis was successful in milk due to the antifouling properties of the device, which is based on surface modification by 3-dithiothreitol propanoic acid (DTTCOOH), a novel antifouling thiol linker. Compared to bare and modified (dithiothreitol (DTT), 11-mercaptoundecanoic acid (MUA), and 1-undecanethiol (UDT)) QCM discs, the aptasensor’s antifouling in milk improved by about 82-96%. The antifouling QCM-based aptansensor’s excellent sensitivity and ability to detect and quantify S. aureus in whole UHT cow’s milk demonstrates that the system is applicable for rapid and efficient analysis of milk safety.

  • Open access
  • 18 Reads
Developing a sensitive method for the electrochemical determination of tetracycline using MB tagged aptamers on gold electrode substrates

In this work, we report the development of electrochemical biosensors for the detection of Tetracycline (TET) using DNA aptamer conjugated with the redox probe Methylene blue (MB) with the following sequence: 5’-MB-CCC CCG GCA GGC CAC GGC TTG GGTTGG TCC CAC TGC GCG-thiol-3’ [1]. The aptamer was immobilized on a gold surface working electrode via chemisorption, utilizing the Au-S bond and the detection of TET was performed by differential pulse voltammetry (DPV) [2]. The binding of TET resulted in conformational changes of the aptamer and as a result, MB became closer to the sensor surface, increasing the current peak. Experiments were performed with different buffers with and without NaCl, at pH=7.6 and 5. The binding buffer, leading to best results was the one with NaCl at pH=7.6, which is also in agreement with Alawad et al. [3]. The limit of detection (LOD) was determined to be 60 nM, which is below the maximum residue limit (MRL) in milk samples. Comparisons between current immunosensor technologies and our aptasensor were also performed.

  • Open access
  • 12 Reads
Molecularly Imprinted Polymers-coated Fluorescent Nanomaterials for Detection of Antibiotic Residues

The contamination of water and food with antibiotics residues poses a severe risk to human health and aquatic environment. The excessive and uncontrolled use of antibiotics is one of the major causes of their presence in the environment. Their continuous consumption willingly or un-willingly can result in severe health issues such as allergy, headache, hypertension, muscles pain, and hormonal dysfunction. Besides these, the development of antimicrobial resistance (AMR) can make the situation more critical. Therefore, advanced analytical approaches over conventional techniques are required to detect antibiotics residues in a facile and cost-effective manner. Present study focuses on detection of ciprofloxacin antibiotic residues via fluorescent sensing probes. Here, different nanostructures were synthesized such as quantum dots (QDs), and their composites. For selective and specific detection of antibiotics, molecular imprinted polymers are coated on the surface of fluorescent nanostructures. The use of MIPs over other biomolecules (such as antibody, enzymes, and others) is highly promising that avoids any kind of pre-treatment of sample. The formation of these MIPs-coated nanostructures is confirmed by different microscopic and spectroscopic techniques. The performance of these nanomaterials and their composites-based fluorescent probes was explored for the detection of antibiotic residues in water samples. The results of fluorescent sensor are consistent with the results of high-performance liquid chromatography (HPLC) that confirms the favorable performance of developed fluorescent sensor for highly sensitive and specific detection of antibiotics in practical applications.

  • Open access
  • 28 Reads
Functionalization of Graphene Oxide for Label-free Electrochemical Detection of Hepatic Cancer Cells

In this study, a graphene oxide (GO) modified screen-printed carbon electrode (SPE) was functionalized with 1-pyrenebutyric acid-N-hydroxy-succinimide ester (PANHS) and conjugated with antibodies for the label-free detection of human hepatoma HepG2 cells (Fig. 1). Using a user-friendly reservoir chamber, the functionalized film was exposed continuously to the cancer cells. The continuous flow is intended to enhance capture of the target cells onto the sensing platform. The response of the biosensor was evaluated using cyclic voltammetry (CV). The preliminary data showed good sensitivity for detection of hepatic cancer cells. The developed biosensor could detect the HepG2 cells from 1 × 103 to 3 × 105 cells/mL range. Thus, it provides a simple tool for electrochemical detection of cancer cells and offers low-cost and disposable sensing platform.

Figure 1. Schematic illustration of the developed electrochemical biosensor for detection of cancer cells (not drawn to scale).

  • Open access
  • 8 Reads
Electrochemistry of freely diffusing mediators in polyelectrolyte membranes used for blood glucose test-strips with a high upper limit of the linear range.

Co-immobilization of low molecular weight mediators and glucose oxidase in polyelectrolyte membranes results in glucose test strips operating in millimolar concentration range. Density and charge of polyelectrolyte membranes formed on the surface of the screen-printed electrodes allow to control the diffusion of mediators. Negatively charged perfluorosulfonated ionomer (PFSI) hampers the diffusion of the commonly used ferricyanide (III) ion, while the hexammine ruthenium (III) cation apparent diffusion coefficient in PFSI membrane remains the same as without the membrane. In contrast to PFSI, electrode modification with positively charged chitosan leads to additional adsorption of potassium hexacyanoferrate on the membrane. Additionally, the rate of mediator leakage from the membrane was found to govern the sensitivity of the resulting biosensors. The leakage rate also depends on the density and charge of the polyelectrolyte and mediator. However, the main advantage of the proposed simple approach of single-step deposition of three-component membrane-forming mixtures on the screen-printed electrodes is the extended upper limit of the linearity: 30-50 mM glucose. Hence, the obtained test strips are suitable for glucose detection in undiluted blood.

  • Open access
  • 10 Reads
Effects of intense hot-spot-specific local fields on fluorescein adsorbed at 3D porous gold architecture: evolution of SERS amplification and photobleaching under resonant illumination

Plasmonic nanostructures with a high density of hot spots, which are characterized by high local electromagnetic fields, are needed to increase the sensitivity of surface-enhanced Raman spectroscopy (SERS) and fluorescence (SEF). These nanostructures can be used in both identifying biological molecules and monitoring photochemical reactions that occur on a metal surface when electromagnetic radiation interacts with these molecules. Such analysis methods are widely used both in medicine and pharmacology.

In this work, using the method of pulsed laser deposition, three-dimensional (3D) porous wedge-shaped arrays of gold nanoparticles were obtained with structural parameters varying along the substrate, such as film thickness, porosity, nanoparticles size, and distance between them . The resulting arrays were close-packed multilayer structures with a varying density of hot spots along the substrate, in which the field intensity enhancement correlates with the nanostructure geometric parameters. Using the SERS of fluorescein molecules adsorbed on the surface of the obtained nanostructures, we studied the dynamics of fluorophore photobleaching under the influence of electromagnetic field. The obtained results provide the information about the highest electric field intensity region in hot spots on the surface of plasmonic nanostructures. We observed the similarity in the evolution of SERS signals and fluorescence in time, which indicated the similarity of the SERS amplification and photobleaching mechanism for both processes. An increase in the photobleaching rate of fluorescein for the structure with the highest enhancement of SERS and fluorescence has been observed.

  • Open access
  • 18 Reads
Combination of on-chip electromembrane extraction with solid phase microextraction using a monoithic nanocomposite as the sorbent

Non-steroidal anti-inflammatory drugs (NSAIDs) are one the most commonly prescribed pain killers. NSAIDs can cause adverse health effects on humans such as gastrointestinal disorder, anemia, agranulocytosis and changes in renal functions. Therefore, it is significant to achieve a suitable technique for accurate measuring these compounds. During the last decades, many studies have focused on development of simple, environmentally friendly and efficient miniaturized sample preparation methods with a high clean up ability. In this way, a lot of extraction techniques such as solid phase microextraction (SPME) and electromembrane extraction (EME) have been employed in remarkable number of investigations. As a result of performing two extraction techniques simultaneously, it could be possible to take advantages of both methods. Hence, extraction recoveries, preconcentration factors and other noteworthy extraction features such as ability to clean up through matrix sample could be improved. In this study, Cu/Cr layered double hydroxid was dispersed in poly (methacrilic acid-coethylene glycol dimethacrylate) (MAA-co-EGDMA) polymerization mixture and in-situ polymerization was performed in acceptor phase channel. Thus, a monolithic composite was formed in the channel. Thereafter, EME-SPME on chip was developed to extract naproxen, diclofenac and mefenamic acid from various complex matrices. Likewise, desorbtion step was performed after electromembrane extraction and eluent was injected into high performance liquid chromatography-UV for separation and determination of the drugs. Effective parameters on extraction efficiency were optimized and under optimum conditions, the limit of detections of the mentioned analytes were 0.1-0.25 ng/mL. Linearity of method was obtained within the range of 0.5-500 ng/mL for naproxen and 1-250 ng/mL for dicofenac and mefenamic acid with coefficients of determination greater than 0.996. Under evaluation of this method, extraction recoveries were obtained in the range of 83.34-90.87% which corresponded to preconcentration factors of 56-61. The precision of the method is suitable with relative standard deviations lower than 4.8%. The method was applied for extraction of the drugs from real saples like as breast milk, urine and plasma; and satisfactory results were obtained.

  • Open access
  • 16 Reads
Gold nanostructure orchestrated electrochemical immunosensor integrated with Antibody-Electroactive Probe Conjugate for rapid detection of SARS-CoV2 antibody

Nanomaterials play a major role in the development of both advanced and conventional diagnostic devices. Gold nanomaterials (Au nanoparticles, nano-flowers, magnetic nanobeads, nano-composites, etc) have been widely reported in various sensing technologies, owing to their distinct morphological, optical, electrical, chemical, and physical properties to improve the overall sensing quality. Apart from improving the interface properties they can also be easily functionalized and conjugated to bio-molecules to act as a reporter in colorimetric sensors. Herein, the presented work reports the fabrication of the electrochemical immunosensor for rapid SARS-CoV2 antibody (CoV2-Ab) detection in the blood serum samples with a linear detection range of 10-100ng/ml, linearity R2=0.96, sensitivity 0.013µA(ng/ml)-1cm-2, LOD=3.59ng/ml and LOQ=11.84ng/ml. For the fabrication of the immunosensor, the electrode base was modified with a bio-polymeric layer and then coated with gold nanostructures to enhance the electron charge transfer. The SARS-CoV2 antibody capturing probe was then immobilized to the electrode to detect different concentrations of SARS-CoV2 antibody. To further enhance the readability, electroactive material tagged secondary antibody was used as the reporter, that also amplifies the electrochemical signal in presence of its mediator. The present immunosensor demonstrates a nano structure-based platform for rapid detection of viral antibodies (SARS-CoV2 Ab) and the concept can be used for the development of rapid sensing platforms for different viruses.

  • Open access
  • 12 Reads

Since the emergence of the COVID-19 pandemic, the SARS-CoV-2 virus has continued to evolve, leading to the emergence of many variants worldwide. The qRT-PCR test of respiratory samples is the current gold standard for detecting SARS-CoV-2; however, this test is costly, time-consuming, and requires highly qualified personnel and instruments, making it unsuitable for rapid on-site analysis. Given the population health risk and social factors in Mexico, it is crucial to detect and track the COVID-19 disease in the Mexican population accurately and in a fast manner. Enzyme-linked aptamer-based biosensors are a fast, inexpensive, and simple method for detecting various targets, including viruses. However, a biosensor based on this technology to detect the SARS-CoV-2 virus has not yet been designed. Therefore, this project aims to develop a lab-on-a-chip enzyme-linked optical aptasensor to detect the virus. As a first step, in this study, three toehold aptamer sequences targeting the Nucleocapsid protein gene were developed and studied in silico. This approach reduces experimental time and cost while providing insights into the binding mechanism, binding affinity, and specificity of aptamers. The assays performed involved obtaining the secondary structures of aptamers with the mFold online server, predicting the 3D modeling structures and potential binding sites with the RNAcomposer modeling server and PyMOL software, performing molecular docking simulations of the target and aptamer, evaluating aptamer stability, and determining binding energies with high accuracy. The results obtained from the in-silico assays suggest that these aptamers are a promising option for detecting the SARS-CoV-2 virus. Further experimental testing is needed to determine whether these sequences can be integrated into a biosensor technology and used to process and test patient samples.

  • Open access
  • 15 Reads

Currently, dietary supplements contain a wide range of non-specific concentrations of testosterone and/or its synthetic analogues, substances that are not permitted and that pose a risk to public health, which puts into perspective the need to evaluate and regulate the composition of these products. The present project proposes as a control tool, the development of a biosensor using aptamers as bio-recognition elements. Aptamer is specific sequence of oligonucleotides with the ability to fold into unique three-dimensional structure that interact with the analyte (testosterone and analogues). In an integral way it is proposed that the aptamers are coupled to gold nanoparticles functioning as a census and signal transduction system conducing to a biosensor with high sensitivity and selectivity, as well as rapid response. In this work, modeling and molecular docking tools were used to evaluate the folding and structural stability of the aptamers. It is essential to carry out the in silico analysis, in a complete way for the bio-recognition system, to evaluate the stability of the proposed aptamers to various variations in the medium, allows to determine the conditions and adaptations necessary for experimental, design and operation of the biosensor. On the other hand, evaluating the affinity and identifying the type of interactions between aptamer and analyte, allows to locate the best candidate, of the proposed aptamers. The stability of a set of nine sequences, with proven interaction towards testosterone, was evaluated under different conditions: folding temperature (8.0 °C, 20 °C and 30 °C), [Na+] (1.0 mm M, 50 mM and 150 mM) and [Mg2+] (1.0 mm M, 2.0 mM, 3.0 mM and 4.0 mM), with Mfold web server, RNA Composer and PyMOL. The affinity and molecular interaction assays were carried out between each of the aptamers and three analytes: testosterone, testosterone undecanoate and androstenedione using Autodock Vina, Chimera, Pymol and Discovery Studio. The results of stability and conformational changes of the aptamers are exposed in a graph of three variables, and it was obtained the aptamers (T6, T5.1 and TESS1) are compatible with the conditions to be run tests and have high affinity for testosterone, whose interactions are constituted mainly by Van der Waals forces, hydrophobic interaction and hydrogen bonds.