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  • Open access
  • 80 Reads
Reduced Graphene Oxide on Screen-Printed Carbon Electrodes as Biosensor for Escherichia coli O157:H7 Detection

Rapid development and deployment of biosensors for pathogen quantification is needed for flood-stricken areas. Drinking-water supplies in flooded areas become a disease threat owing to mixture with sewage discharge. In such exigent conditions, water is filtered through a microfiltration technique that is moderately effective in removing bacteria and non-effective in removing viruses. Quantification of pathogens using biochemical techniques does not provide real-time data and requires transportation of water samples to laboratories for analysis. However, transportation services are severely affected during flooding, and hence on-site testing may be the only option. WHO specifies that diagnostic devices are to be made ASSURED – affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free, and deliverable to end-users. To fulfil ASSURED criteria, we developed a low-cost electrochemical biosensor integrated with an Arduino® microprocessor encased in a portable platform for E. coli O157:H7 quantification. The working electrode (WE) of a screen-printed carbon electrode (SPCE) was modified with reduced graphene oxide (rGO). Rabbit serum IgG, a non-specific antibody towards E. coli O157:H7, was immobilized on the WE. We tested the specificity of E. coli O157:H7 binding to IgG/rGO/SPCEs and rGO/SPCEs at 4 to 4 × 108 CFU/ml. We chose a potential range of -0.35 to 0.07 in LSV to compare the current values at different concentrations. At -0.14V, IgG/rGO/SPCEs distinguished E. coli concentrations of 4 and 4 × 108 CFU/ml with current value of 17 µA and 1 µA, respectively. However, the IgG/rGO/SPCE does not distinguish between E. coli concentration of 4 × 107 and 4 × 108 CFU/ml. The rGO/SPCE does not distinguish E. coli concentration of 4 and 4 × 108 CFU/ml, as evidenced by almost similar current values in both concentrations. This study indicates the potential of non-specific antibodies for E. coli O157:H7 quantification during water-quality monitoring in flooded areas.

  • Open access
  • 83 Reads
Fabrication of An All-Solid-State Ammonium Ion–Selective Electrode by a Two-Step Process Using Cyclic Voltammetry

Ammonium ion (NH4+) is one of the indicators of water quality; high ammonium concentration [NH4+] in water can cause eutrophication, affect aquatic biota, and cause cell death in the central nervous system of human beings. However, current ion-selective electrodes used for water-quality monitoring are bulky, require frequent calibration owing to membrane fouling, and cannot be integrated into mobile sensor platforms. We fabricated an all-solid-state ion-selective electrode (AS-NISE) for ammonium ion using a two-step process; the first step is electropolymerization deposition on the electrode using a solution of 3,4-ethylenedioxythiophene (EDOT), sodium polystyrene sulfonate (NaPSS), and lithium perchlorate (LiCLO4), resulting in a solid-state transducer on screen-printed carbon electrodes (SPCEs), and the second is electropolymerization deposition of EDOT, NaPSS, and o-phenylenediamine (o-PD) as ammonium ion–selective membrane (NISM) on top of the transducer. The electropolymerization deposition of the transducer and the NISM were simply achieved by cyclic voltammetry (CV) with potential from 0.0 V to 0.8 V and 50 mVs-1 scan rates. The fabricated NH4+ISE can detect [NH4+] as low as 5.7×10-5 M, with slope of 61.9 mV/decade (R2>0.99) and a linear range from 10-3 M to 1 M. These preliminary results provide an initial insight into the applicability of the simple two-steps fabrication process of NH4+ ISEs for scaling-up purposes, with the ability for miniaturization and integration into a mobile sensor platform.

  • Open access
  • 143 Reads
Tailoring the Electrochemical and Morphological Properties of Electropolymerized and Dropcast rGO:PSS-PEDOT:PSS Transducers for Ion-Selective Sensors

Fabrication of ion-selective sensors for continuousmeasurement in fluids depends on understanding the electrochemical and morphological properties of transducers. Electropolymerized nanomaterials essentially offer stable transducers that can reduce measurement drifts. This study aims to elucidate the electrochemical and morphological characteristics of electropolymerized reduced graphene oxide stabilized in polystyrenesulfonate and poly(3,4-ethylenedioxythiophene): polystyrenesulfonate composites on screen-printed carbon electrodes (rGO:PSS-PEDOT:PSS/SPCEs) using scanning electron microscopy (SEM) and cyclic voltammetry (CV) in 0.1 M ferricyanide (Fe(CN)64/3-). We fabricated the rGO:PSS-PEDOT:PSS/SPCEs by two different techniques: electropolymerization deposition (EPD) and drop-casting (DC). Results revealed smaller peak-to-peak potential separation (∆Ep) of 360 mV for EPD rGO:PSS-PEDOT:PSS/SPCEs, compared to 510 mV for the DC rGO:PSS-PEDOT:PSS/SPCEs. A smaller ∆Ep indicates higher reversibility and faster electron-transfer rate at the electrode-analyte interface. SEM results showed EPD rGO:PSS-PEDOT:PSS/SPCEs have the roughest surface among electrodes; homogeneous globular structures with diameter range of 1.45.3 μ m covered the electrode surface. In terms of electrode integrity in fluids, cracks can be seen on the surface of DC PEDOT:PSS/SPCEs after undergoing CV in 0.1 M Fe(CN)64/3-, whereas rGO:PSS-PEDOT:PSS/SPCEs for both deposition methods maintained their integrity. Globular structures of rGO:PSS-PEDOT:PSS using EPD methods remained after undergoing CV. The results suggest that EPD serves as a potential method to fabricate a stable transducer for ion-selective sensing. This study aims to elucidate performance of nanocomposites via EPD methods, to develop stable ion-selective sensors for physiological and environmental applications.

  • Open access
  • 92 Reads
Peptide-based biosensor for express diagnostics of coronavirus respiratory infections
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At the end of 2019 the first reports appeared of a new coronavirus and WHO announced a pandemic of COVID-19. To date, the new coronavirus, now called SARS-CoV-2, has infected more than 25, 000,000 people and killed about 900, 000 worldwide. It is extremely important to develop means for express diagnostics to ensure prompt action to limit the spread of infection. One of the diagnostic approaches is the detection of viral particles in swabs. This approach can be realized using biosensor with specific ligands, based on peptide molecules complementary to surface viral proteins. The concept of the so-called Systems of Conjugated Ionic-Hydrogen Bonds (abbreviated - SSIVS, CIHBS) implemented in the Protein-3D computer program, was applied to analyze the spatial structures of the bonds between the SARS-CoV-2 spike protein and the ACE-2 receptor, in order to reveal the perspective peptide sequences. There are two clearly marked areas of contact of the spike with the cell receptor - upper and lower, which are visualized in the SSIVS form, and the complex formed at this site is strong enough to ensure its attachment to the coronavirus spike and can compete for binding with the ACE-2 receptor. Two peptides were developed that form a spatial structure complementary to the coronavirus spike: of 8 (No 1) and of 15 (No 2) amino acid residues. The peptides were covalently bound to biochip platforms via neutral linkers to form two sites, including: peptide No1, No 2. The third site has a neutral hydrophilic surface to serve as a reference. The platform was integrated with microfluidic channel and was used as a flow through device. The detection of bound viral particles was carried out using UV excitation and direct registration of viral proteins fluorescence. The preliminary laboratory tests demonstrated the efficiency of the biosensor.

  • Open access
  • 91 Reads
Microcentrifuge tubes as disposable immunoelectrochemical cells for the on-site detection of GFAP, biomarker of hemorrhagic stroke
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Nowadays, stroke is the leading cause of mortality worldwide. Hemorrhagic (HS) and ischemic stroke (IS) have different pathophysiology and treatment. While there is a treatment for IS that reverses its most devastating effects, this does not happen with HS, and treatment for IS could even worsen HS. Therefore, differential diagnosis between IS and HS in the acute stage is an important challenge. Current diagnostic of acute stroke relies on neuroimaging techniques that provide valuable information but not always are readily available. Therefore, the development of analytical tools for rapid and on-site detection of biomarkers capable of differentiate between the types of stroke could enhance the optimally manage of patients. Glial fibrillary acidic protein (GFAP) is considered one of the acute HS biomarkers; its levels in serum are increased in patients with HS in comparison with IS patients.

In this work, a sandwich-type immunoassay for the determination of GFAP was wholly performed inside a microcentrifuge tube immobilizing the capture antibody on its bottom. The electrodes needed for the detection are included in the cap in a simple and innovative strategy that allows to follow an all-in-one procedure. Thus, the cap is drilled with three stainless-steel pins that act as electrodes. With this design, when the tube is closed the head of the pins is inside and thus, once the immunoassay is finished, the tube is turned upside down allowing the electrochemical detection in the same tube.

The good precision of this tube-based electrochemical cell makes possible to obtain a point-of-need device with huge potential for analytical applications that require rapid and on-site response. In the case of GFAP determination, this device, combined with a handheld potentiostat, would be of great interest for its use in out-of-hospital sites to help determine the cause of the stroke in the acute stage.

  • Open access
  • 57 Reads
New Au based nano/microstructures for the development of a new aptasensor for oxytetracycline
Published: 02 November 2020 by MDPI in The 1st International Electronic Conference on Biosensors session Posters

Oxytetracycline (OXT) is an important with widespread use antibiotic. But, like in the case of other antibiotics, its overuse fuels the rise of the problem of antibiotic resistance.

In this context, there is a clear need for the development of new, fast and sensitive analytical methods capable of performing in field analysis. Electrochemical sensors, like electrochemical aptasensors are viable solution for this [1].

The aim of our work was the developemnt of an aptasensor for OXT, using as a starting platform carbon-based screen printed electrodes (C-SPE), modified with Au-based nano/microstructures. These platforms were imagined and used due to their relatively lower cost compared to Au-based screen printed electrodes, and also to test the influence of the arhitecture of the Au nano/microstructures on the analytical performance of the aptasensor. As a biomimetic element, we used a thiolated deoxyribonucleic acid (DNA) aptamer, labelled with ferrocene.

The Au-based platforms and all the steps involved in the aptasensor development were electrochemically characterized, both indirectly using ferrocene and Ferro/ferricyanide redox probes and directly based on the electrochemical signal of the Au structures and on the signal of the labelled aptamer. The arhitecture of the Au structures were investigated using the scanning electron microscopy technique.

In conclusion, the creation and characterization of new Au-based nano/microstructures on C-SPE was carried out. The resulting analytical platforms were selected based on their influence on the immobilization of the aptamer and on the analytical performance, one of them being chosen as the starting point for the development of the final aptasensor.

Acknowledgements: This work was supported by a grant of the Romanian National Authority for Scientific Research and Innovation, CCCDI-UEFISCDI, project number ERANET-RUS-PLUS-PLASMON-ELECTROLIGHT/46/2018, within PNCDI III. GS thanks UMF for the internal grant number 2461/71/17.01.2020. OH thanks to the Romanian Ministry of Education and Research, CNCS - UEFISCDI, project number PN-III-P1-1.1-PD-2019-0631, within PNCDI III.

  1. Y. Li, J. Tian, T. Yuan, P. Wang, and J. Lu, Sensors Actuators, B Chem., vol. 240, pp. 785–792, 2017, doi: 10.1016/j.snb.2016.09.042.
  • Open access
  • 100 Reads
Metallic pins as electrodes in low-cost (bio)electroanalytical devices
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The increasing demand of fast and on-site information has grown the interest in developing simple and portable analytical devices that provide reliable responses. Electrochemical biosensors fit perfectly with these purposes because of the combination of their selectivity and ease-of-use with the simplicity, low-cost and facility of miniaturization of the electrochemical transducers. Moreover, the growing attention that has attracted the construction of do-it-yourself electronic devices in the last years has spread the use of common and mass-produced materials for the development of analytical devices. In this work, we present the use of gold-plated pins from standard connections and stainless-steel pins from needlework as electrodes in (bio)electroanalytical platforms.

In the first example, the gold-plated pins, which acted as reference (RE) and counter (CE) electrodes, were combined with a paper-based working electrode (WE). This platform showed good analytical features and it was used for developing an enzymatic glucose sensor.

The performance of stainless-steel pins as electrodes was also demonstrated constructing an enzymatic glucose sensor in which a transparency sheet was used as substrate and the pin acting as WE was modified with carbon ink. In a different design, the high versatility of the pins as electrodes allowed to insert them in a micropipette tip to obtain a system able to take the sample and perform electrochemical measurements in the same tip. This device was used for determining anionic surfactants using methylene blue as indicator.

All these innovative low-cost platforms showed good features for analytical determinations and those metallic commercial pins have demonstrated to be extremely useful for the design of interesting and simple electrochemical devices.

  • Open access
  • 71 Reads
Realization of enhanced evanescent field long period fiber grating near turn around point for label-free immunosensing

A Long period Fiber Grating (LPFG) with maximum enhancement of evanescent field has been designed and fabricated, along with theoretical modeling, by working near the turn-around point (TAP) of lowest order symmetric cladding mode (LP0,2 cladding mode). The LPFG was fabricated using point by point inscription technique and it was characterized in term of surrounding refractive index (SRI) within the range of 1.333 to 1.3335 using a thermostated flow-cell. This closed cell, made of poly(methyl methacrylate) (PMMA), was designed for better handling of the sensor, because during the fabrication process the diameter of the LPFG was reduced up to ~ 20 µm by chemical etching, for the maximum enhancement of evanescent field. The sensitivity of dual peak resonance of the LP0,2 cladding mode near TAP was measured and resulted to be ~ 8751 nm/SRIU with a resolution of the order of 10-5 RIU. The sensor was further used for the label-free immunosensing application by the implementation of Immunoglobulin G (IgG)/ anti- Immunoglobulin G (anti-IgG) bioassay on the grating region inside the thermostated closed flow cell and for real effectiveness along with the confirmation of the specificity, we perform the negative control test of the sensor using a complex matrix consisting of human serum.

  • Open access
  • 129 Reads
Microfluidic assay based on G-quadruplex aptamer for nucleolin detection in prostate cancer
, , , , , , , , , ,
Published: 02 November 2020 by MDPI in The 1st International Electronic Conference on Biosensors session Posters

Prostate cancer (PCa) diagnosis methods lack specificity and sensitivity or are extremely invasive. Thus, the fast, simple, reliable and accurate detection of low-grade PCa biomarkers could have a great clinical impact, saving lives and reducing patient uncertainty.

Studies reported the overexpression of the protein nucleolin (NCL) on the cell surface of PCa cells and can be considered as a cancer-selective target. NCL binds specifically G-rich sequences, namely DNA G-quadruplex (G4) aptamers like AS1411, which can be modified and act as a molecular beacon (MB) for detection.

In the present work, we have designed a microfluidic platform for the detection of NCL using a modified AS1411 aptamer beacon (AS1411-N5). When the MB recognizes NCL, it is prompted to undergo a conformational change, separating the fluorophore from the quencher and restoring fluorescence.

Firstly, we started to characterize structurally the MB. Circular dichroism and Nuclear Magnetic Resonance spectroscopies indicated that the MB forms a parallel G4 structure. The affinity with NCL was determined fluorometric titration. Localization of MB was assessed in the PCa cell line (PC-3), further demonstrating the ability of the MB to recognize cell-surface NCL. To assess the clinically relevant NCL concentration of human PCa, ELISA assay was performed using peripheral blood mononuclear cells of PCa patients. Finally, the microfluidic assays showed the capacity of the microfluidic device in recognizing NCL in complexed samples like spiked human plasma.

Concluding, it is here demonstrated that the microfluidic platform based on the AS1411-N5 could be used as a specific and selective approach to detect NCL in biological samples and contribute to the early-stage diagnosis of PCa.

  • Open access
  • 68 Reads
Acoustic biosensor for discrimination of pathogens according to Gram-principle

The microacoustic methods of biomedical analysis, implemented on piezoelectric crystals and ceramics are becoming increasingly perspective due to potential of integration, as functional elements of biosensors. An important stage in diagnostics of infectious diseases is the identification of pathogens. One of possible applications of such sensor is an alternative to time and labor consuming Gram method of discriminating bacteria according to composition of their cell walls. Thus, bacteria which in a procedure of Gram staining, do not decolored after application of dye solution, are classified as Gram-positive (G(+)). They are surrounded with the thick peptidoglycan layer, pulpy and dampening acoustic waves. While Gram-negative (G(–)) bacteria, which acquire red color in Gram procedure, are covered with thin and springy layer, demonstrating resonance effects when interact with acoustic fields. Thus, G(+) and G(–), which differently colored in Gram procedure are also react differently to external acoustic field: for G(–) bacteria, this is a sharp decrease in the Q-factor of the "resonator-suspension" system and a shift of the resonance curve to lower frequencies. While for G(+) bacteria, although a certain shift of the resonance curve was also observed, but the bandwidth of resonance curve practically did not change. This effect was studied for L. acidophilus (G(+)) and E. coli (G(–)) bacilli with quarts resonators of 4 MHz, 5 MHz and 10 MHz. The biosensor was testing using Lactobacillus fermentum, E. coli M-17, Bifidobacterium bifidum, Burkholderia cepacia, Staphylococcus aureus. At this stage it has been demonstrated that the method is particularly effective for discriminating bacteria of the similar size, such as, for example cocci. The discrimination of Gram factor for cocci and bacilli was less accurate and needs further studies for selection of precise resonance frequencies.