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  • Open access
  • 43 Reads
Optimization procedures for development of SERS-based lateral flow assay for high sensitive detection of Troponin I
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Cardiovascular disease (CVD) is identified as the leading cause of death because of heart disease, stroke and other chronic diseases. The early and rapid detection of Troponin I, a cardiovascular disease biomarker, has an imperative role to prevent high risk of death. A lateral flow immunoassay (LFA) for detecting Troponin I quickly and effectively would provide yes/no answers with visual assessment, but not adequate to monitor the early stage of CVD. The Surface-Enhanced Raman Scattering (SERS) technique, offering highly sensitive and quantitative analysis, is integrated with LFA in order to measure Troponin I in a highly specific manner. Although the application of LFA is a modern technique, the development of SERS-based LFA is not straightforward. This research discusses three optimization procedures to develop SERS-based LFA for high sensitivity detection of Troponin I: (a) optimizing gold nanoparticle sizes (30, 50, 80, 100 nm) for SERS quantitative assay on Troponin I LFA; (b) investigating LFA components and fluid flow time to recognize Troponin I with SERS performance; and (c) evaluating different laser wavelengths and laser power for SERS-based LFA for the analysis of Troponin I. In a SERS-based LFA, these parameters are fundamental for augmenting sensitivity and detection limit. The SERS-based LFA became more sensitive than visual detection with naked eyes for quantitative quantification of Troponin I after optimization. In addition, the discussed procedures may offer advantages for the development of SERS-based LFA to detect various biomarkers in a highly sensitive manner.

  • Open access
  • 15 Reads
Engineering next generation bioluminescent Ca2+ sensors through directed evolution

The use of genetically encoded fluorescent sensors for calcium ion (Ca2+) has revolutionized neuroscience research by allowing for the recording of dozens of neurons at the single-cell level in living animals. However, fluorescence imaging has some limitations such as the need for excitation light, which can result in high auto-fluorescent background and phototoxicity. In contrast, bioluminescent sensors using luciferase do not require excitation light, making them ideal for non-invasive deep tissue imaging in mammals. Our lab has previously developed a bioluminescent Ca2+ sensor CaMBI to image Ca2+ activity in the mouse liver (Oh, et al. Nat Chem Biol 2019), but its responsiveness to Ca2+ changes was suboptimal. To improve the performance of this sensor, we applied directed evolution to screen for genetic variants with increased responsiveness. Through several rounds of evolution, we identified variants with more than five times improved responsiveness in vitro. We characterized the improved sensors in culture cell lines and dissociated rat neurons and confirmed that they exhibited higher sensitivity to changes in intracellular Ca2+ levels compared their progenitor. These optimized Ca2+ sensors have the potential for non-invasive imaging of Ca2+ activity in vivo, particularly in the brain.

  • Open access
  • 12 Reads
A cell-based bioelectric biosensor for Salmonella spp. detection in food

The prevalence of foodborne diseases has significantly increased in the last decades, causing numerous hospitalizations and deaths, as well as money loss in the agri-food sector and food supply chain worldwide. Several factors such as increased population density, rapid urbanization, and globalization have contributed to this increase. The standard analyses currently used for bacteria detection have significant limitations with the most important being their long procedural time that can be crucial for foodborne outbreaks. Hence, there is an imperative need for new, accurate techniques that can provide fast results for pathogens presence to withdraw the contaminated products from the markets before their distribution to consumers, thus preventing pathogen dispersal and human infection. In this study, we developed a biosensor system able to perform robust and accurate detection of Salmonella spp. in meat products after a 3-minute analysis. To achieve this, we used a portable device developed by EMBIO Diagnostics called B.EL.D (Bio Electric Diagnostics) and a cell-based biosensor technology (BERA). Biosensors were created using monoclonal anti-Salmonella spp. mouse antibodies and tests were conducted in cured meat and raw meat samples, while five different protocols with different incubation periods were evaluated for their validity. Results indicated that the new method could detect the pathogen within 24 hours after a 3-minute analysis and discriminate samples with and without Salmonella with high accuracy (86.1%). The method’s sensitivity, specificity, and positive and negative predictive values ranged from 80 to 90.5%, while the limit of detection was determined to be as low as 10 CFU g-1 in all food substrates.

  • Open access
  • 17 Reads
Assessment of the freshness of fish and poultry meat by fast protein and metabolite liquid chromatography using a new optical sensor

Fresh fish and poultry meat are in high demand on the market: poultry, mainly chicken, is the second most consumed and the most affordable meat product in the world. Fish consumptions varies greatly across regions but in some countries, seafood is the main source of abundant and affordable macro- and micronutrients.
Meat and, especially, fish are highly perishable products: methods and equipment for rapid, objective, and reliable assessing the freshness of fish and meat are crucial for the food industry. Generally recognized reference techniques such as total volatile basic nitrogen (TVB-N), volatile fatty acids (VFA), high pressure liquid chromatography (HPLC), mass spectrometry, or nuclear magnetic resonance (NMR) spectroscopy are time-consuming and require expensive and complex equipment.
We developed a novel chromatographic optical sensor with a deep UV LED photometric detection (255–265 nm) for rapid assessment of meat and fish freshness based on determination of the relative content of adenosine triphosphate (ATP) metabolites. The sensor has a simple and compact design, and relatively low cost; sample preparation and processing of a chromatogram takes less than 30 min.
The sensor was tested on Amur (farmed freshwater fish) and rooster meat, obtained from a local farmer. The samples were kept refrigerated at +4 C°, measurements were taken daily during a 14 days period. All chromatograms show two peaks: the first one is responsible for proteins; the second broad post-protein band is formed due to the overlapping of individual peaks of ATP and its metabolites. As fish and poultry meat are stored, ATP is converted into metabolites with lower molecular weight, which is reflected in the chromatograms – the elution time for the second peak increases. It was shown that this time can be directly associated with the freshness status of a product. As expected, poultry meat showed better storage stability and freshness retention compared to Amur fish.

  • Open access
  • 76 Reads
Hydrogel-coated nanonet-based field-effect transistors for SARS-CoV-2 spike protein detection in high ionic strength samples

The SARS-CoV-2 pandemic has triggered many studies worldwide in the area of biosensors, leading to innovative approaches for the quantitative assessment of COVID-19. Nanostructured field-effect transistor (FET) are one type of the devices shown to be ultrasensitive for virus determination. FETs can be used as transducers to analyze changes in electrical current caused by the bonding of viral molecules to the surface of the semiconducting nanomaterial layer of the FETs1. Although nano-transistors require simple setups amenable to be miniaturized for point-of-care diagnostic of COVID 19, this type of sensors usually have limited sensitivity in biological fluids. The reason behind is the shortened screening length in the presence of high ionic strength solutions2. In the frame of this study, we propose a methodology consisting on the FET surface modification with a hydrogel based on the star-shaped polyethylene glycol (starPEG), which hosts specific antibodies against SARS-CoV-2 spike protein in its porous structure. The deposition of the hydrogel increases the effective Debye length, preserving the biosensor’s sensitivity. We demonstrate the capability of silicon nanonet-based FETs to detect the viral antigens and cultured viral particles in phosphate-buffered saline (PBS) as well as in human purified saliva. Finally, we discriminated positive and negative patients’ nasopharyngeal swab samples.

  1. Ibarlucea B, Fawzul Akbar T, Kim K, et al. Ultrasensitive detection of Ebola matrix protein in a memristor mode. Nano Res. 2018;11(2):1057-1068. doi:10.1007/s12274-017-1720-2
  2. Stern E, Wagner R, Sigworth FJ, Breaker R, Fahmy TM, Reed MA. Importance of the Debye Screening Length on Nanowire Field Effect Transistor Sensors. Nano Lett. 2007;7(11):3405-3409. doi:10.1021/nl071792z
  • Open access
  • 13 Reads
Preliminary results of the development of a DNA hybridization-based biosensor for the detection of milk adulteration using gold interdigitated electrodes.

Milk is a widely consumed product and its adulteration is not only widespread but also very dangerous. This study aimed to develop a biosensor for the detection of milk adulteration using DNA hybridization. The advantages of biosensors over traditional laboratory methods, such as their speed, ease of use and cost-effectiveness, are combined with the sensitivity of DNA hybridization. A capacitive biosensor was developed using interdigitated gold electrodes on paper substrate, which were modified with specific oligonucleotides for cow mitochondrial DNA that served as the biorecognition element. The methodology relies on the measurement of changes in capacitance due to DNA hybridization. Preliminary results are presented, showing the ability of this biosensor to detect bovine DNA in goat milk with high sensitivity and specificity. The results show that this biosensor has the potential to be a low-cost, easy-to-perform, and fast method for the detection of milk adulteration. This biosensor technology is a promising development for the detection of milk adulteration and can help to ensure the safety and quality of milk products.

  • Open access
  • 7 Reads
MD Average of Vibrational Spectra of Nucleotides in a SERS Sensor Simulation with varying number of Au nanoparticles

Applying the surface-enhanced Raman scattering (SERS) method to detect bioactive molecules such as DNA, proteins, and drugs has significant potential for structure-sensitive nondestructive chemical analysis. The SERS discrimination of single molecule oligomers in DNA, microRNA, and proteins has attracted wide attention due to the possibility of developing new sensing technology. The collected signal’s sensitivity has the level of detection of single oligomers, which can be compared with the simulation results corresponding to the sensor structure. We investigate the averaging method of the individual bond spectra for DNA nucleotides in the ring part of the pyrimidine (6-ring) and purine (6-5-ring) bases to form vibrational spectra obtained by molecular dynamics (MD) simulation. The system consists of the Au nanoparticles (from 1 to 4 NP assay) attached to a graphene sheet at the edge of the nanopore that localizes in the nanopore nucleotide interaction & spectral enhancement. The nucleotide translocation velocity set at 0.025 m/s compares with the experimental range. The vibrational spectra ring average has been tested for adenine and guanine with close correspondence (in the 500-1700 cm-1 range) to the experimental Raman & SERS spectra and extended to cytosine and thymine nucleotides. We also modified the number of the Au nanoparticles from 1 NP to 4 identical NPs to evaluate the influence of the interaction on the MD transient spectra. The variations of mode frequencies and amplitudes due to the number of Au NPs in bond spectra, as well as ring averages, mark the corresponding Au – nucleotide interactions and are considered to be used as training sets for machine learning methods.

  • Open access
  • 12 Reads
Cell classification based on artificial intelligence analysis of cell images in microfluidic chip

Biological cells are micro-particles with certain size, shape and state. Most biological cells have complex, irregular appearance and internal structure, organic or chemical composition and biological activity. At present, the classification of biological cells includes traditional optical microscope, fluorescent straining, flow cytometry or PCR, etc. However, these methods suffer several drawbacks such as low sensitivity, expensive and bulky instruments and/or complex processes. With the rapid development of computer algorithms, artificial intelligence (AI) starts to be applied in cell classification, which is simple and does not need any labelling reagent. The main problems of cell classification based on AI are focused on the influence of image background, cell aggregation, small size and lack of enough training samples. We developed a low-cost, multi-classification, label-free and high-precision method for cell classification, which combines microfluidic technology with deep learning algorism together. The recognition of states of red blood cells (RBCs) was selected as the typical example to demonstrate the feasibility of the method. The microfluidic channel is designed to effectively and controllably solve the problem of cell overlap, which has severe negative impact on the identification of cells. The object detection model based on YOLOv5 in the deep learning algorism is optimized and used to recognize multiple RBCs simultaneously in the whole field of view, so as to classify them into six morphological subcategories and count the numbers in each subgroup. The blood quality can be evaluated by calculating the morphology index according to the numbers of cells in subgroups. The validation of the method is verified by evaluating three blood samples stored for 1 week, 3 weeks and 6 weeks, which have distinct morphology index differences. This method has the merit of cell identification in a wide channel, no need for single cell alignment as the image cytometry does and it has potential applications to the classifications of biological cells with different morphologies.

  • Open access
  • 13 Reads
Micro-weighing based biosensor with using an adaptive interferometry

Mass of a macroscopic object is easily measured by a suitable balance. However, this approach becomes inapplicable if it is necessary to measure the masses of micro- and nano-objects such as biomolecules, cells, viral particles. Alternative approach for mass measurement is based on using of micromechanical resonator as an inertial balance which oscillation frequency depends by added mass value. There are various ways to measure the micro-oscillators resonant frequency: optical, piezoelectric transducers, electrostatic, etc. Optical frequency measurement methods are the most versatile. Measurements by optical methods make it possible to measure vibrations in a non-contact way. In this work, an adaptive holographic interferometer with implemented for measurement of oscillator frequency. A silicon micro-cantilever with dimensions 125×30×2.5 µm3, which plays the role of the sensing element, is mounted on the positioning system. The cantilever was fixed on a piezoquartz plate to excite vibrations by sinusoidal electrical signal. Cantilever vibrations were measured by the adaptive interferometer. The interferometer object wave being reflected from the cantilever propagates through the semi-insulating photorefractive CdTe crystal, where it is mixed with the reference wave. Wave coupling provides phase-to-intensity transformation in the object wave. Changes in the intensity of the object wave were recorded using a photodetector connected to an analyzer of the spectrum of electrical signals. The recorded spectrum of the electrical signal was the amplitude-frequency spectrum of the cantilever oscillations. Cantilever-type microoscillators, as a rule, have a high QF of oscillations, therefore they always have a clearly defined resonant frequency, which for the cantilever used in this work was 253442±23 Hz. In the experiment, mass of absorbed molecules of Bovine serum albumin (BSA) was measured on different concentration of water solution. It is shown that biosensor is able to measure concentration of BSA water solution with concentration in 0.2 mg/ml.

  • Open access
  • 9 Reads
Chemical Transformation of Typical Biological Recognition Elements in Reactions with Nanosized Targets: A Study of Glutathione Coated Silver Nanoparticles

The study of the interaction between biologically active molecules and plasmonic nanoparticles has attracted an increasing interest due to their wide application in biosensors and biomedical diagnostics. In addition to their practical applications in medicine and pharmacology they are also used in fundamental research carried out in studying processes occurred in biological systems. Glutathione is a complexing agent that plays a key role in the functioning of a living cell. This molecule is a tripeptide one having the structure as L-glutamine-L-cysteine-glycine (γ-Glu-Cys-Gly; GSH) and performs intra-and intercellular transport of metal ions in biological systems. Thiol (“reduced”) glutathione and disulphide (“oxidized”) glutathione often act as actuators of molecular nano-switches that activate, regulate or inhibit the activity of enzymes as a response to the presence of different levels of reactive species, such as ROS or electrophilic organic metabolites.

Gold nanoparticles are often used as transducers of nanosized biosensors, since they have the optical and chemical properties necessary for sensory applications and make it possible to form a sensitive layer with the desired functionality on its surface using various molecular recognition species, in particular, glutathione molecule. Due to the presence of thiol groups in this molecule, direct irreversible immobilization of glutathione on the gold surface occurs at the room temperature without any additional activation procedures. Such coatings are in demand in medical preparations. For example, the coating of gold nanoparticles (AuNPs) with glutathione led to a new class of inorganic nanoscale carriers excreted by the kidneys with high resistance to serum protein adsorption, which can deliver drugs to or directly effectively affect the tumour under the influence of external factors (electromagnetic fields).

Silver nanoparticles also have attracted an increasing interest to sensor applications due to different spectral range, typically the narrower plasmon resonance band compared to gold nanoparticles, different surface chemistry and agglomeration behaviour. For example, conjugates of glutathione with silver NPs are characterized by the presence of circular dichroism, which indicates the instability of the attachment of glutathione molecules to the silver surface. Features of the behaviour of glutathione molecules on the surface of silver nanoparticles determined the purpose of this work, which was focused on studying the sequential evolution of glutathione on the surface of silver nanoparticles.

Despite the apparent simplicity of the procedure for modifying silver nanoparticles with glutathione, spectrophotometry and TEM data indicate that the interaction of glutathione molecules with silver nanoparticles is a rather complex chain of chemical transformations. At the initial stage, as in the case of gold nanoparticles, clustering of nanoparticles is observed. However, then, the formed clusters are destroyed with the formation of an insoluble precipitate. In this case, the disappearance of the plasmon resonance band and the appearance of additional absorption are observed that indicates the transformation of silver nanoparticles into silver sulphide. This compound is characterized by extremely low solubility in aqueous media and is not any more a source of silver ions, which are toxic to living cells. So, the use of silver nanoparticles in an environment where glutathione is present reduces the toxic effect of silver nanoparticles themselves. It should be noted that the formation of metal sulphides as detoxification forms of some thiol-binding metals has been recognized in several organisms, from microorganisms and plants to mammals.