Recently, surface-enhanced Raman scattering (SERS) nanoprobes have shown tremendous potential in oncological imaging owing to the high sensitivity and specificity of their fingerprint-like spectra. As current Raman scanners rely on a slow, point-by-point spectrum acquisition, there is an unmet need for faster imaging in real-time. The development of single-molecule resolution in the tip-enhanced and surface-enhanced Raman spectroscopy (TERS & SERS) promotes experimental work on DNA and protein identification by the above methods and approaches a single oligomer resolution that leads to its use as a nanobiosensor. However, a weak signal requires multiple acquisitions of the averaged spectra to enhance the signal, and information on the molecular conformation and interaction is erased. The enhancer Au clusters of known geometry, size, and position relative to the measured molecule will help to build spectral libraries for single spectral signal prediction.

As the simulation molecular dynamic (MD) model of a sensing system, we study vibrational spectra of the cytosine nucleotide in the dynamic interaction with the Au₂₀ nanoparticles (NP) that can be later attached to graphene nanopore. We define nucleotide and NP localization and study the influence of interaction on the spectral modes of both the nucleotide and NP, as we had seen in the case of the nucleotide-graphene nanopore. The spectral maps of the nucleotides were built in FF2/FF3 potential. Fourier transfer of the density of states (DOS) was performed to obtain the spectra of various bonds in reaction coordinates for DNA nucleotides at a numerical resolution 20 to 40 cm^-1. The pyramid-shaped Au₂₀ NP was optimized by ab initio DFT GGA and relaxed by the MD calculation with the EAM potential with Δt=0.1fs. Spectral maps of the Au NP were acquired for each atom. The frequencies that can serve as markers of the corresponding Au – nucleotide interaction have been evaluated.

Morbus Parkinson belongs to the most frequent disorders of the central nervous system. The loss of dopamine-producing cells causes a lack of the neurotransmitter dopamine in the brain. This deficiency results in progressive movement disorders - typical symptoms of Parkinson‘s disease (PD). Due to the central role of dopamine in the catecholamine metabolism numerous enzymes are regulating its concentration, for example the catechol-O-methyl transferase (COMT). The treatment of PD is based on the regulation of the COMT activity by administration of COMT inhibitors (entacapone, tolcapone). For a personalized medication the observed change of the PD symptoms should be complemented by direct measurements of enzyme activity.

Here, the determination of the enzyme substrate dopamine was performed by differential pulse voltammetry (DPV). By the applied electrode material fluorine doped tin oxide (FTO) a clear discrimination between the substrate dopamine and the conversion product methoxytyramine is possible. A linear dependency of the dopamine oxidation signal on the concentration in the range of the maximum reaction rate of the COMT and a high signal stability during consecutive measurements allow a reliable sensor construction. Even though none of the individual essential assay components induces a current signal at the FTO electrode, in the complete activity assay the dopamine oxidation signal was influenced by each added ingredient. After adjustments of the DPV potential range and changes of the assay composition a reproducible determination of dopamine concentrations in the assay can be achieved. Investigations with agarose-bound COMT demonstrate clearly the dependency of the voltammetric dopamine signal on the substrate incubation time with the enzyme. Furthermore, the signal change correlates clearly to the activity of bound enzyme, indicating that enzyme action can be followed by electrochemical means and demonstrating the suitability of FTO as electrode material for activity detection of the COMT via DPV.

For effective treatment, it is critical to determine a correct diagnosis when an individual is encountering an infection. Various cutting-edge technologies have been designed and used in diagnosis in the last recent years.
Aptamers are short single-stranded DNA or RNA oligonucleotides that bind to a specific target molecule, reproducing antibodies' role while improving the functional effect. Aptamers are obtained via an in vitro chemical process named as systematic evolution of ligands by exponential enrichment (SELEX). The SELEX technology achieved high improvements using magnetic-beads for the aptamer-target molecule selection.

For over half a century, glycopeptide antibiotics have been used as a key weapon in the fight against bacterial infections. Vancomycin is a powerful glycopeptide antibiotic, which can be toxic in high doses to renal and auditive systems, but also at low doses can cause hypersensitivity reactions. Even if microbiological resistance to vancomycin is not commonly developed, clinical treatment failures oftentimes occur with long-term susceptibility for infection remission [1]. In this way, it is critical to measure with as high as possible accuracy the concentration of vancomycin from biological and environmental samples, having the aim to improve the patient compliance to treatment and to overcome the multi-antibiotic resistance issue.
This poster presents our current progress in the selection of a new aptamer through magnetic beads-based SELEX technology that will be further explored for its potential application in the detection of vancomycin in wastewaters.

Acknowledgments:

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.

References:

[1] K. Hiramatsu, Y. Kayayama, et al., Journal of Global Antimicrobial Resistance, (2014), 2, 213–224.

Early detection of foodborne pathogens is significant for ensuring food safety. Nowadays, the detection of pathogens found in food can take up to 72 hours and it might take a week to confirm positive sample. While standardized methods give test results in a shorter period of time, the reoccurring costs for each measurement are high. That is why it is necessary to develop technology that will be cheap, fast, simple and accurate enough. Biosensors in combination with aptamers offer such possibilities.

This work is focused on the development and testing of a biosensor based on DNA aptamers for detection of pathogenic bacteria Listeria innocua using the method of quartz crystal microbalances (QCM). The aptasensor was formed on the surface of an oscillating piezo crystal, whose frequency was affected by deposited mass. An aptamer specific to the genus Listeria spp was used for detection of pathogen, which includes 16 subspecies. 3 subspecies are excluded as their antigen structure differs from other species (L. murrayi, L. grayi, L. ivanovii).

We found that when Listeria innocua cells interacted with an aptamer specific for Listeria spp. Addition of the pathogens at the QCM transducer modified by aptamers resulted in decrease of the resonant frequency in concentration depending manner. We also confirmed the specificity of the aptamer used for Listeria innocua, as neglected response of the sensor took place for E. coli for which Listeria spp. has some partial antigens identical and thus can cause cross-reactions in serological tests. The developed aptasensor showed promising sensitivity and specificity for real-time detection of Listeria innocua, with a detection time of 30 min. The achieved limit of detection was approximately 1.6 x 10³ CFU / ml.

Acknowledgement

This work was funded by the European Union, project Horizon 2020, Marie Skłodowska-Curie, contract number 690898 and was also supported by the scientific grant agency VEGA, project number: 1/0419/20.

In this work we focus on the electro-chemical detection of bacteria in a nitrocellulose (NC) membrane with interdigital electrodes (IDE) through impedance spectroscopy.

Paper-based sensors are inexpensive, simple and portable tools for environmental monitoring, in particular, in resource-limited settings. They mostly rely on specific bioreceptors and nanoparticles for colorimetric pathogen detection, and perform limited semi-quantitative measurements. Electrical biosensors offer opportunities for quantitative pathogen detection, usually with planar electrodes that sense bacteria attachment to their surface through changes in interfacial impedimetric properties. In particular, impedance spectroscopy allows for impedimetric sensing over a wide frequency range. However, they show limited sensitivity due to low bacteria number attached onto the planar biosensor.

To overcome these limitations, we innovate classic biosensing techniques by depositing planar electrodes directly on the NC, a paper derivative, with the aim of sensing electrical properties on the whole volume of the NC membrane. We take advantage of the natural capillarity of NC to bring bacteria solutions at the testing zone.

The complete sensing device is fabricated and characterized. An analytical model is established and validated by analyzing the conductivity changes of the NC volume, seen by the IDE and caused by the bacteria presence inside the porous NC.

To ensure high specificity and binding capacity to bacteria, the NC membrane is functionalized with phage endolysin cell-wall binding domain (CBD) as bioreceptor, deriving from the endolysin encoded by Deep-Blue, a bacteriophage targeting B. thuringiensis.

A proof of concept of the proposed biosensor is substantiated: 10⁸ CFU/ml of B. thuringiensis are detected in DI water.

Through easy and appropriate modification of the biointerface, this affordable and sensitive biosensor creates opportunities in applications that need frequent and rapid pathogen detection, such as the detection of E. coli in drinking water; or various viruses, which may prove particularly useful regarding COVID-19 pandemic.

Breast cancer is the leading cancer site for women with 2 million new yearly infections and more than half a million dead worldwide. Human Epidermal Receptor 2 (HER2) is a prominent breast cancer biomarker that indicates aggressive cancer and is often associated with a bad prognosis and low survival rates. However, current detection methods for HER2 are often time-consuming, expensive, and require a high level of expertise.

Biosensors are devices that turn biological interaction into a readable electronic signal; they are known for their high specificity and selectivity for low concentration, as well as their low cost and ease of use, thus making them a better alternative to traditional methods. Also, Saliva is becoming a better alternative for blood for the detection of biomarkers due to its non-invasive collection in large quantities with simple collection methods with a richness in disease biomarkers including HER2. Thus this project aims to develop a label-free, low cost, electrochemical biosensor for the detection of HER2 in saliva.

This was done by first depositing diazonium salt onto a screen-printed electrode (SPE) through cyclic voltammetry, then immobilizing anti-HER2 antibodies on the activated SPE using the EDC/NHS protocol [1]. HER2 biomarker concentrations were detected using electrochemical impedance spectroscopy inside a microfluidic system. The biosensor showed a higher linear detection of HER2 (Y= 0.0062 X + 0.1066/ R² = 0.9909) in its physiological concentration range of 5 and 40 pg/mL when compared to other interference proteins: Epidermal Growth Factor Receptor (Y= 0.0016 X + 0.0188/ R² = 0.8072) and Human Epidermal Receptor 3 (Y= (0.0035 X + 0.0225/ R² = 0.1302). The biosensor was then used to detect 10 pg/mL of HER2 concentration in real saliva using the standard addition methods (Y = 0.0118 X + 0.1282/ R² = 0.9876).

Reference

1. Abrao Nemeir, I. et al. Impedimetric label-free detection of salivary EGFR on screen printed electrode. Integr. Cancer Sci. Ther. 2019, 6, 1–3.

Oncological diseases belong to the most serious diseases with high mortality. The most common cancer in children is acute lymphoblastic leukemia (ALL). It is a cancer of the blood-forming tissues characterized by a large increase in the numbers of leukocytes in the circulation or bone marrow. The prognosis of positive treatment increases with early diagnosis of this disease. It is therefore important to develop diagnostic methods that will be able to detect this disease in early stage. One of the option can be non-invasive diagnostics using the biosensors based on nucleic acid - aptamers. Aptamers are characterised by unique properties such as high specificity and nonimmunogenicity. Aptamer recognizes the surface markers on the membrane of cancer cells with the high binding affinity. Biosensors based on aptamers with redox markers are among the most sensitive experimental tools of this type.

In this contribution, we report the development of optimizing redox-labeled electrochemical aptasensor for the detection of Jurkat leukemia cells. The aptamers specific to the protein tyrosine kinase 7 (PTK7), the important membrane protein cancer marker that is overexpressed in Jurkat cells were used. We compared the sensitivity of aptasensors for aptamers modified either by methylene blue (MB) and ferrocene (Fc), respectively. Both aptasensors were tested in the presence of Jurkat cells at concentration range 50-5000 cells/ml using differential pulse voltammetry. In both cases the comparable sensitivity was obtained with limit of detection of approx. 200 cells/mL according to 3S/N rule.

Acknowledgement

This work was funded by Science Agency VEGA, project No. 1/0419/20.

Mono- and multi-metal nanoparticles (MNPs), thanks to their unique and tunable features, still fascinate the analytical sciences, still tracing a witness major breakthrough in nanotechnology-based colorimetric sensing. Starting from the widespread use of colloidal MNPs in sensing and biosensing as nanoplasmonic tag or catalyst to finally end to the MNPs immobilization onto solid substrates. Here, ELISA microplates and micro-Tips decorated with adhesive and plasmonic-active polymeric films embodying gold/silver nanostructures will be presented. The support surfaces were initially modified with self-assembled gold nanoparticles on polydopamine films. Subsequently, the AuNPs-decored film was doped with catechin leading to the formation of a uniform silver plasmonic nano-network in the presence of Ag⁺. The bimetallic nanoarchitecture on ELISA microplate was exploited to assess oxidants and free radicals (H₂O₂, sodium hypochlorite, ABTS•⁺, etc.) using the LSPR decrease caused by silver nano-network etching as analytical signal. For the studied oxidants, the system allows differentiating the oxidants' ability with useful limits of detections, throughout a simple photometric/optical reading. Moreover, taking advantage of a similar strategy a nanodecored lab-on-a-tip was developed, the device speed-up the 4-nitrophenol reduction retaining the performances for more than 10 consecutive measures. In this case, the improved catalytic activity of the nanohybrid film (gold/silver) was demonstrated, resulting higher compared with the monometallic films also allowing a fast 4-nitrophenol colorimetric monitoring. In this presentation, a bimetallic adhesive robust and storable film able to modify different supports is proposed, the supports modified with such film result able to determine and monitor different molecules of analytical and biological interest without the needing of additional reagents. The film fabrication results versatile and potentially usable onto different supports and materials allowing tailor-nano decorations, proposing itself as a new and useful analytical read-out system.

Silver nanoclusters (AgNCs) generated on DNA-templates belong to a new class of fluorescent tags showing excellent brightness, photostability as well as biocompatibility. Thus, AgNCs-DNA have been applied in various applications, from the detection of DNA/RNA, environmental monitoring to bioimaging and cancer therapy. In this work, we report fluorescent AgNCs synthesized using two 1,3-diaza-2-oxophenothiazine (tC)-modified oligonucleotides that contain RET-related sequence CCCCGCCCCGCCCCGCCCCA. The communication compares the absorption and emission properties of the obtained systems with silver nanoclusters synthesized on the unmodified oligonucleotide. First, we showed the optimal conditions for AgNCs-DNA synthesis on three DNA templates: (1) RET20 with the sequence 5'-CCC CGC CCC GCC CCG CCC CA-3'; (2) 19tC with the sequence 5'-CCC CGC CCC GCC CCG CCC tCA-3'; (3) 14tC with the sequence 5'-CCC CGC CCC GCC CtCG CCC CA-3'. Next, the silver nanoclusters were characterized by UV/Vis absorption, fluorescence and circular dichroism spectroscopy. Silver nanoclusters RET19tC-AgNCs and RET14tC-AgNCs indicated the several times higher fluorescence intensities in the long-wave emission spectra as compared to RET20-AgNCs. Moreover, silver nanoclusters on tC-modified oligonucleotides showed the higher stability over time. The possibility of using of the silver nanoclusters RET19tC-AgNCs and RET14tC-AgNCs for monitoring pH changes would be also demonstrated.

NASA Ames Research Center is the leader in developing autonomous nanosatellites or CubeSats to address strategic knowledge gaps about the effects of space travel on biological organisms, including GeneSat, PharmaSat, and EcAMSat. Now that NASA has set its sights on human exploration in deep space, such missions require significant technological and biomedical countermeasures to protect astronauts from chronic radiation exposure. CubeSats can inform these countermeasures by querying relevant space environments with model organisms and/or biosensors.

BioSentinel will be the first interplanetary CubeSat to study the biological response to space radiation outside low Earth orbit in almost 50 years. BioSentinel is an autonomous platform able to support biology and to investigate the effects of space radiation on a model organism in interplanetary deep space. It will fly onboard Artemis-1, from which it will be deployed on a lunar fly-by trajectory and into a heliocentric orbit.

BioSentinel, a 6U CubeSat (1U = 10-cm cube), will measure the DNA damage response to ambient space radiation in a model organism, which will be compared to information provided by an onboard radiation spectrometer and to data obtained on the ISS and on Earth. Even though the primary objective of the mission is to develop an autonomous spacecraft capable of conducting biological experiments in deep space, the 4U BioSensor science payload contained within the free-flyer is an adaptable instrument that can perform bio measurements with different microorganisms and in multiple space environments, including the ISS, lunar gateway, and on the surface of the Moon. Thus, nanosatellites like BioSentinel can be used to study the effects of both reduced gravity and space radiation and can house different organisms to answer specific science questions. In addition to their flexibility, nanosatellites also provide a low-cost alternative to more complex and larger missions, and require minimal crew support, if any.