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  • Open access
  • 80 Reads
Label-free peptide-based biosensor for express detection of protein markers of acute cardiovascular conditions in biological fluids
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Acute cardiovascular conditions require prompt assistance, which depends on a timely and accurate diagnosis. This could be achieved by using biosensor systems based on peptide aptamers capable of selectively binding protein markers of diseases. In this work, a label-free biosensor system based on fluorimetric registration of the formation of a "peptide aptamer - target protein" complex is considered. It comprises a microfluidic subsystem integrated with arrays of sites with immobilized peptide aptamers, coupled with optical detection system. The clinical sample of the whole blood is loaded into the inlet basin, where the cells are separated and plasma flows into the microfluidic channel for analysis. Peptide aptamers were created using the molecular complement search technique based on the search for systems of conjugated ion-hydrogen bonds in the three-dimensional structures of target proteins. The technology for manufacturing a microfluidic chip is a combination of thick-film and photolithography technologies based on the SU-8 photoresist, for which the relief and surface morphology have been studied. The composition of the biochip layers is selected in such a way that ultraviolet light with a wavelength of 280 nm passes through inlet window, excites fluorescence inside the channel, which passes through the glass window, which absorbs UV-light. This wavelength accounts for the maximum absorption of aromatic amino acids - tyrosine and tryptophan. In this case, one of the last layers is a luminophore layer for re-emission of protein fluorescence as a visible light. The reading platform includes a 280nm LED, a video sensor, tooling, 3D-printed PLA tooling, and software for processing and analyzing the received signal.

  • Open access
  • 105 Reads
Development of electrochemical DNA detection methods to measure circulating tumour DNA for enhanced diagnosis and monitoring cancer

Liquid biopsies are becoming an increasingly important potential replacement for existing biopsy procedures which can be invasive, painful and compromised by tumour heterogeneity. This paper reports a simple electrochemical approach tailored towards point of care cancer detection and treatment monitoring from biofluids using a label free detection strategy. The mutations under test were the KRAS G12D and KRAS G13D mutations which are both important in the development and progression of many human cancers and whose presence correlates with poor outcomes. These common circulating tumour markers were investigated in clinical samples and amplified by standard and specialist PCR methodologies for subsequent electrochemical detection.
Following pre-treatment of the sensor to give a clean surface, DNA probes developed specifically for detection of the KRAS G12D and G13D mutations were immobilized onto low cost carbon electrodes using diazonium chemistry and EDC/NHS coupling. Following functionalisation of the sensor it was possible to sensitively and specifically detect mutant KRAS G12D and G13D PCR product from cancer patients against a background of wild type KRAS DNA from the representative sample. Our findings give rise to the basis of a simple and very low cost system for measuring ctDNA biomarkers in patient samples. The current time to result of the system is 4.5 hours with considerable scope for optimisation and already compares favourably to the UK National Health Service biopsy service where patients can wait weeks for their result.
The paper will report the technical developments we have made in the production of clean carbon surfaces for functionlisation, show the assay performance data for KRAS G12D and G13D with a range of PCR systems and demonstrate the potential for measuring response to treatment offered by the system.

  • Open access
  • 148 Reads
Detection of cocaine and cathinones by electrochemical fingerprinting using nanomaterials
Published: 02 November 2020 by MDPI in The 1st International Electronic Conference on Biosensors session Posters

Illicit drugs use and abuse remains an increasing challenge for worldwide authorities and, therefore, it is important to have accurate methods to detect them in seized samples, biological fluids and wastewaters. They are recently classified as the latest group of emerging pollutants as their consumption increased tremendously in recent years.

Nanomaterials have gained much attention over the last decade in the development of sensors for a myriad of applications. The applicability of these nanomaterials, functionalized or not, significantly increases and are therefore highly suitable for use in the detection of drugs of abuse.

We have assessed the suitability of various nanoplatforms for the electrochemical detection of illicit drugs, such as graphene, singled-walled carbon nanotubes, multi-walled carbon nanotubes functionalized with platinum nanoparticles, gold nanoparticles and platinum nanoparticles. Graphene and singled-walled carbon nanotubes were drop-casted onto graphite screen printed electrodes and left to dry at room temperature. Multi-walled carbon nanotubes were functionalized with platinum nanoparticles using a simple and efficient chemical process, in which carboxylic acid-functionalized multi-walled carbon nanotubes were mixed with chloroplatinic acid in a mixture of ethylene glycol and water to result in platinum-functionalized multi-walled carbon nanotubes. The functionalized multiwalled carbon nanotubes were drop-casted onto graphite screen printed electrodes. Gold and platinum nanoparticles were deposited by electrodeposition from a solution of chloroauric acid and chloroplatinic acid, respectively, by cyclic voltammetry.

The electrochemical fingerprints of cocaine and cathinones (such as mephedrone, alpha-polyvinylpirolidone, methylmethcatinone, chloroethcatinone, chloromethcathinone, etylone) were elucidated on the above-mentioned nanoplatforms. Square wave voltammetry was performed as a high-performance electrochemical method. This allowed for the sensitive and selective (class selectivity) of the investigated illicit drugs.

  • Open access
  • 78 Reads
Comparison of the performance of two aptamers on a colorimetric assay for the quantification of FB1
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Fumonisin B1, a mycotoxin commonly produced by Fusarium verticillioides and classified as group 2B hazard, has been quantified in a variety of food products. After the first reported aptamer (96 nt ss-DNA) for the highly specific molecular recognition of FB1, only 28 aptamer-based biosensors have been published, of which 50% quantified fluorescent signals. A critical point, yet commonly overlooked during the design of aptasensors, is the selection of the binding buffer. In this work, a simplified colorimetric assay was designed by incubating a folded aptamer with FB1, and the subsequent addition of gold nanoparticles (AuNP). The change in the aggregation profile of AuNP by a 40 nt aptamer or a 96 nt aptamer, was tested after addition of the target molecule under different buffer conditions. The incubation with Tris-HCl and MgCl2, exhibited the most favorable performances from the 40-mer and 96-mer, respectively; however contrary to previous publications, the short sequence was not specific to FB1 as it also portrayed signal suppression with other mycotoxins. On the other hand, the assay developed with the longest aptamer was specific to FB1 and comparable to other sensitive aptasensors with a limit of detection of 3 ng/mL (A650/520 ratio) and 2 ng/mL (absolute peak area). Contrary to other mechanisms, particle stabilization was triggered by an increasing target concentration. Additionally, the application of more complex methods such as asymmetric flow field-flow fractionation (AF4) allowed the analysis of diverse signals (peak area (λ), refractive index, average diameter), to increase the sensitivity up to the pg/mL level. To our knowledge, this is the first colorimetric study reporting the sole application of an unmodified aptamer for the detection of FB1, thus avoiding the application of complementary sequences and allowing the formation of a complex AuNP-aptamer-FB1 caused by MgCl2.

  • Open access
  • 106 Reads
Medical H2S sensing with gold and platinum surface modified ZnO nanowire ChemFETs
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Over the past decade, numerous publications highlighted the medical role of hydrogen sulfide (H2S) for therapeutic applications and early diagnostics, traced in medical breath analysis e.g. with the “electronic nose” approach. It was shown that abnormal endogenous H2S concentration levels in exhaled breath samples can be linked to airway inflammation in asthma patients, and hence H2S functions as a potent biomarker.

In this work, we investigate the catalytic effects of gold (Au) and platinum (Pt) nanoparticle layer deposition on highly sensitive zinc oxide (ZnO) nanowires (NW) used for selective H2S detection in the sub-ppm region. High quality pristine ZnO NWs are grown by high temperature chemical vapor deposition (CVD) and vapor liquid solid growth (VLS) on silicon with a thin Au layer acting as a growth catalyst. The surface modification of pristine ZnO NWs was modified by systematical magnetron sputtering of discontinuous Au and Pt nanoparticle layers of 1 – 6 nm thickness. Resistive gas sensors based on the gas sensing mechanism of a chemical field effect transistor (ChemFET) with open gate, which is formed by hundreds of parallel aligned pristine, Au modified or Pt modified ZnO NWs, were measured towards H2S diluted in dry nitrogen (N2) or in dry synthetic air at room temperature. Gas sensing results allow for a first understanding of largely improved signal-to-noise ratio and response due to the catalytic effects of metal deposition on ZnO NW surface. Controlled application of optimized ZnO NW growth and metal catalyst deposition show a clear enhancement of response towards 1 ppm H2S from initial 15 % with pristine ZnO to 5000 % with ZnO after 5 nm Au deposition and hence greatly lower the limit of detection.

  • Open access
  • 81 Reads
Comparison of optical and gravimetric methods for detection of chymotrypsin

Proteases play important role in various biological processes as well as in the dairy industry. In this work we were focused on detection of chymotrypsin which is an important protease in human digestion.

We performed comparative analysis of detection chymotrypsin activity by two methods - optical and acoustical. In the first case we used gold nanoparticles (AuNPs), modified with β-casein. We characterized the changes in absorption spectra after modification by β-casein and 1-mercaptohexanol (MCH). This modification protected AuNPs from aggregation. Addition of chymotrypsin resulted in cleavage of β-casein, which caused changes in absorption spectra due to formation of AuNPs aggregates. The absorption spectra were measured every 15 minutes up to one hour after chymotrypsin addition, which allowed us to monitor the kinetics of the β-casein cleavage. By measuring the absorption maximum, change of its position and change in absorbance, it was possible to construct calibration curve and to determine limit of detection (LOD). This value was 0.21 nM. In the second case we detected chymotrypsin by means of the acoustic shear mode method (TSM). β-casein was immobilized on the surface of the TSM transducer (AT cut piezo-crystal). Addition of chymotrypsin resulted in an increase of resonance frequency, f, and a decrease of motional resistance, Rm, indicating cleavage of short peptide fragments from β-casein. In this case the LOD = 3.1 nM. Thus, both methods represent effective tool for study of the chymotrypsin activity. The assay based on AuNPs, however, allowed for better sensitivity and easier operation.


A portion of this research was conducted at the Center for Nanophase Materials Sciences, project No. CNMS2018-293. This work was funded under European Union’s Horizon 2020 research and innovation program through the Marie Skłodowska-Curie grant agreement No 690898 and by Science Agency VEGA, project No. 1/0419/20.

  • Open access
  • 164 Reads
“Green” nanozymes: synthesis, characterization and application in amperometric (bio)sensors

Nanozymes (NZs) are catalytically active nanomaterials, which have enzyme-like activity, but possess increased stability and greater availability due to simpler preparation technologies. NZs as nanoscale artificial enzymes possess various catalytic specificities as oxidoreductases, such as peroxidase, catalase, laccase and others, as well as hydrolases, proteases, endonucleases, DNA-ases, NO synthases, etc. Many NZs exhibit dual- or multienzyme mimetic activity. NZs as stable low-cost mimetics of natural enzymes have a high potential for application in different branches of biotechnology, including scientific investigations, industry and ecology. NZs can be applied in medicine as diagnostic tools and components of therapeutic drugs. Since NZs have high catalytic activity and chemical and biological stability, they are very promising in construction of biosensors and biofuel cells. For these reasons, the search for simple methods of synthesis and characterization of different NZs is a very important and actual problem.

The “green” synthesis of Prussian blue analogous as peroxidase-like NZs using oxido-reductases is described in this study. The obtained green-synthesized hexacyanoferrates (gHCF) of transition metals were characterized by structure, size, composition, catalytic properties, electro-mediator activities and substrate specificity. Copper hexacyanoferrate (gCuHCF) was studied in more detail. When immobilized on a graphite electrode (GE), gCuHCF under special conditions of pH and tension, gives amperometric signals on hydrogen peroxide and therefore can be used as a peroxidase mimetic in oxidase-based biosensors. Under other conditions, gCuHCF/GE reacts to other analytes. We propose that gHCF of transition metals synthesized via enzymes may become prospect platforms for construction of multi-functional amperometric (bio)sensors.

Funding: This work was partially funded by NAS of Ukraine (The program “Smart sensor devices and technologies”), by the Ministry of Education and Science of Ukraine (Ukrainian- Lithuanian R&D Project # М/20 - 2020; 12.08.2020) and by the Research Authority of the Ariel University, Israel.

  • Open access
  • 82 Reads
Electronic Eye for identification of Tequila samples

The growing interest of spirits producers to evaluate the authenticity, quality and safety of products has motivated the development of efficient, portable, and low-cost analytical systems capable of monitoring these products' characteristics. This new perspective on analytical instrumentation has been focused on using bio-inspired systems that base their operation on the emulation of human senses to determine food characteristics, for example, color, shape, or size. In particular, the electronic eye (EE) has been designed to mimic human vision and analyze the color and some other attributes related to the sample's appearance.

The present work shows the potential of this bio-inspired system, based on a spectrometry system capable of detecting different Tequila samples. Tequila is a Mexican spirit with a protected designation of origin (POD) made from the blue agave plant, whose worldwide consumption ranks fourth after whiskey, vodka and rum.

The reported system analyzes small volumes of Tequila Reposado and Blanco by calculating samples' absorbance, using a low cost and portable instrumentation employing a CCD camera.

The absorbance imaging method consisted of exciting samples with light passes through an 8MP camera connected to a Raspberry Pi Card. The camera's image data are analyzed using Matlab 2018b to be represented in Red, Green and Blue (RGB) components for each pixel, in order to get an approximation of the absorbance and the surface color index (Isc) associated with sample concentration.

Using the developed EE, it was possible to identify seven different kinds and brands of Tequila. From the obtained results, it is possible to observe that the average absorbance of the Tequila Reposado was greater than the absorbance of the Tequila Blanco. Otherwise, with the Isc, the Tequila Reposado's color index is lower concerning the Tequila Blanco. Finally, the EE allows the identification of Tequila samples with reproducibility and repeatability

  • Open access
  • 80 Reads
Electrokinetically Assisted Paper-based DNA Concentration for Enhanced qPCR Sensing

Paper-based microfluidics have gained widespread attention for use as low-cost microfluidic diagnostic devices in low-resource settings. However, variability in fluid transport due to evaporation and lack of reproducibility with processing real-world samples limits their commercial potential and widespread adoption. We have developed a novel fabrication method to address these challenges. This approach, known as “Microfluidic Pressure in Paper” (μPiP), combines thin laminating PDMS membranes and precision laser-cut paper microfluidic structures to produce devices that are low-cost, scalable, and exhibit controllable and reproducible fluid flow dynamics similar to conventional microfluidic devices. We present a new μPiP DNA sample preparation and processing device that reduces the number of sample preparation steps and improves sensitivity of the quantitative polymerase chain reaction (qPCR) by electrophoretically separating and concentrating nucleic acids (NA’s) continuously on paper.

Our device was assembled using two different microfluidic paper channels: one with a larger pore (25 microns) size for bulk fluid transport and another with a smaller pore size (11 microns) for electrophoretic sample concentration. These two paper types were aligned and laminated within PDMS sheets, and integrated with adhesive copper tape electrodes. A solution containing a custom DNA sequence was introduced into the large pore size paper channel using a low-cost pressure system and a DC voltage was applied to the copper tape to electrophoretically deflect the solution containing NA’s into the paper channel with the smaller pore size. Samples were collected from both DNA enriched and depleted channels and analyzed using qPCR. Our results demonstrate the ability to use these paper devices to process and concentrate nucleic acids. Our concentration device has the potential to reduce the number of sample preparation steps and to improve qPCR sensitivity, which has immediate applications in disease diagnostics, microbial contamination, and public health monitoring.

  • Open access
  • 59 Reads
Detection of sub-nanomolar concentration of trypsin by thickness-shear mode (TSM) acoustic wave biosensor

Determination of protease activity is very important for disease diagnosis, drug development, and assuring quality and safety of dairy products. Therefore, the development of low-cost methods for assessing protease activity is critically essential. Here, we demonstrate acoustic wave-based biosensor operated in the thickness-shear mode (TSM) enables low-cost detection of protease activity in a real-time mode. The TSM sensor was based on a protein substrate (PS) β-casein immobilized on a piezoelectric quartz crystal electrode. The β-casein layer was immobilized onto a gold surface by carboxylate terminated self-assembled monolayer (SAM) of 11-mercaptoundecanoic acid (MUA). Activation of the carboxylic acid terminal was performed by reaction of a mixture of water-soluble N-(3-Dimethylaminopropyl)-N0-ethylcarbodiimide (EDC) and N–Hydroxysuccinimide (NHS) on the electrode surfaces. We demonstrated that β-casein can form stable assembly on a piezoelectric quartz crystal electrode. After enzymatic reaction with trypsin, it cleaved the surface-bound β-casein substrate, which increased the frequency of crystal in a sigmoidal manner. Trypsin was detected in the range of concentrations 1–50 nM. The limit of detection was 0.2 nM. Initial reaction rates measured at different enzyme concentrations have been used to construct a calibration curve. In consideration of results obtained we believe that the TSM biosensor is a useful tool for protease analysis.