Developments in nanoparticles for use in biosensors to assess food safety and quality

The following will provide an overview on how advances in nanoparticle technology have contributed towards developing biosensors to screen for safety and quality markers associated with foods. The novel properties of nanoparticles will be described and how such characteristics have been exploited in sensor design will be provided. All the biosensor formats were initially developed for the health care sector to meet the demand for point-of-care diagnostics. As a consequence, research has been directed towards miniaturization thereby reducing the sample volume to nanolitres. However, the needs of the food sector are very different which may ultimately limit commercial application of nanoparticle based nanosensors. © 2014 Elsevier Ltd

A cellulose-based bioassay for the colorimetric detection of pathogen DNA

Cellulose-paper-based colorimetric bioassays may be used at the point of sampling without sophisticated equipment. This study reports the development of a colorimetric bioassay based on cellulose that can detect pathogen DNA. The detection was based on covalently attached single-stranded DNA probes and visual analysis. A cellulose surface functionalized with tosyl groups was prepared by the N,N-dimethylacetamide-lithium chloride method. Tosylation of cellulose was confirmed by scanning electron microscopy, Fourier transform infrared spectroscopy and elemental analysis. Sulfhydryl-modified oligonucleotide probes complementary to a segment of the DNA sequence IS6110 of Mycobacterium tuberculosis were covalently immobilized on the tosylated cellulose. On hybridization of biotin-labelled DNA oligonucleotides with these probes, a colorimetric signal was obtained with streptavidin-conjugated horseradish peroxidase catalysing the oxidation of tetramethylbenzamidine by H2O2. The colour intensity was significantly reduced when the bioassay was subjected to DNA oligonucleotide of randomized base composition. Initial experiments have shown a sensitivity of 0.1 μM. A high probe immobilization efficiency (more than 90 %) was observed with a detection limit of 0.1 μM, corresponding to an absolute amount of 10 pmol. The detection of M. tuberculosis DNA was demonstrated using this technique coupled with PCR for biotinylation of the DNA. This work shows the potential use of tosylated cellulose as the basis for point-of-sampling bioassays.Peer reviewedFinal Accepted Versio

Developing nucleic acid-based electrical detection systems

Development of nucleic acid-based detection systems is the main focus of many research groups and high technology companies. The enormous work done in this field is particularly due to the broad versatility and variety of these sensing devices. From optical to electrical systems, from label-dependent to label-free approaches, from single to multi-analyte and array formats, this wide range of possibilities makes the research field very diversified and competitive. New challenges and requirements for an ideal detector suitable for nucleic acid analysis include high sensitivity and high specificity protocol that can be completed in a relatively short time offering at the same time low detection limit. Moreover, systems that can be miniaturized and automated present a significant advantage over conventional technology, especially if detection is needed in the field. Electrical system technology for nucleic acid-based detection is an enabling mode for making miniaturized to micro- and nanometer scale bio-monitoring devices via the fusion of modern micro- and nanofabrication technology and molecular biotechnology. The electrical biosensors that rely on the conversion of the Watson-Crick base-pair recognition event into a useful electrical signal are advancing rapidly, and recently are receiving much attention as a valuable tool for microbial pathogen detection. Pathogens may pose a serious threat to humans, animal and plants, thus their detection and analysis is a significant element of public health. Although different conventional methods for detection of pathogenic microorganisms and their toxins exist and are currently being applied, improvements of molecular-based detection methodologies have changed these traditional detection techniques and introduced a new era of rapid, miniaturized and automated electrical chip detection technologies into pathogen identification sector. In this review some developments and current directions in nucleic acid-based electrical detection are discussed

Rapid and Sensitive Detection of Foodborne Pathogens Using Bio-Nanocomposites Functionalized Electrochemical Immunosensor with Dielectrophoretic Attraction.

Ph.D. Thesis. University of Hawaiʻi at Mānoa 2017

Electrochemical and Optical Biosensors in Medical Applications

Analysis of many biochemical processes is of great significance for clinical, biological, food, environmental as well as bioterror applications. But, exchanging of the biochemical information to kind of electronic signal is a defiance due to connecting an electronic tool directly to a biological surrounding. Electrochemical detection instrument due to its advantageous to analyze the subject of a biological sample has a great potential in conversion of a biochemical occurrence to an electronic signal

Nanopipettes as Monitoring Probes for the Single Living Cell: State of the Art and Future Directions in Molecular Biology.

Examining the behavior of a single cell within its natural environment is valuable for understanding both the biological processes that control the function of cells and how injury or disease lead to pathological change of their function. Single-cell analysis can reveal information regarding the causes of genetic changes, and it can contribute to studies on the molecular basis of cell transformation and proliferation. By contrast, whole tissue biopsies can only yield information on a statistical average of several processes occurring in a population of different cells. Electrowetting within a nanopipette provides a nanobiopsy platform for the extraction of cellular material from single living cells. Additionally, functionalized nanopipette sensing probes can differentiate analytes based on their size, shape or charge density, making the technology uniquely suited to sensing changes in single-cell dynamics. In this review, we highlight the potential of nanopipette technology as a non-destructive analytical tool to monitor single living cells, with particular attention to integration into applications in molecular biology

Label-free detection of tuberculosis DNA with capacitive field-effect biosensors

The detection of pathogens from sample material from infected patients is the basis on which a considerable medical diagnosis can be made. Pathogens can be clearly identified based on their genomic material (DNA). A large number of different DNA detection methods with individual advantages and disadvantages have been established. If such methods should be used for certain applications, e.g. for point-of-care measurements, there are a number of requirements which should be considered: A measurement must be performed very fast, inexpensive, simple and reliable. It has been shown that label-free detection principles in particular the field-effect based detection-methods, meet the given requirements. In this thesis, the development of a new measuring method for the detection of DNA (with sequences from Mycobacterium tuberculosis) using a field-effect sensor, is described. The electrolyte-insulator-semiconductor (EIS) structure was selected as the basis for the sensor chip because it has the simplest structure of all field-effect sensors and is inexpensive to manufacture. EIS sensors are capacitive structures that can be read out using an impedance analyzer. The measured value is directly related to the surface potential of the sensor. If the DNA, which is negatively charged in solution, is brought close to the sensor surface, this causes a change in the surface potential via a change in the charge situation on the chip surface. This change in potential can be read out with the help of the EIS sensors: The detection method is based on the detection of a hybridization event on the sensor surface. The surface is modified with a probe single-strand DNA (ssDNA) which has a known sequence that is complementary to the ssDNA that is intended to be detected. As soon as the target ssDNA reaches the surface, a hybridization can occur, whereby a signal shift can be measured. The signal shift is caused by the additional negative charge of the hybridized target DNA molecules. In the case of non-complementary target DNA (ncDNA), there is no hybridization and the signal remains constant. The immobilization of the catcher ssDNA was carried out with by a surface modification using positively-charged polyelectrolyte (poly (allylamine hydrochloride), PAH). Compared to other immobilization strategies that are described in the literature, the capture ssDNA binds adsorptively to the sensor surface, which simplifies the preparation and can be carried out quickly and cheaply. The main topics of this thesis cover the selection of the sensor layout (EIS sensor with SiO2 as surface oxide), the selection and optimization of the surface modification (using PAH), the verification of the forming of double-stranded DNA and the evaluation of the measurement data acquisition by means of capacitive measurements. Due to the adsorptive binding, the DNA strands likely lie flat on the sensor surface. This means that the negative charge of the DNA is located closely to the surface, which means that a high measurement signal can be recorded. The developed protocol was also used with light-addressable potentiometric sensors (LAPS). LAPS are structurally very similar to EIS sensors and have the advantage that they can detect changes in the surface potential in a spatially resolved manner. This makes it possible, for example, to arrange an array so that several DNA experiments can be carried out simultaneously on one chip. However, the measurement setup is more complex because of the necessity of a light source. The measurement of the DNA hybridization on the sensor surface was realized by using the developed method: PAH/ssDNA-modified EIS chips were brought into contact with cDNA solutions. Measurable surface potential changes could show that the hybridization was successful. In direct comparison with experiments where ncDNA was applied to the modified sensor, signal differences of about 11 times higher were measured for cDNA than for ncDNA. The developed method also allows a very simple reuse of the chip by just a repeating of the modification steps on an already used chip. This reusability of the sensors was investigated by performing up to five repetitive surface modification and DNA attachment experiments sequentially with just one chip. A steady decrease in the sensor signal could be observed after each additional layer (PAH or DNA); however, this observation is related to the Debye screening effect. Finally, the developed biosensor was used to detect PCR-amplified cDNA. A detection of the target cDNA was successful and significant, although the additional (interfering) substances in the solution, that were necessary for the PCR process (enzymes, etc.), disturbed the measurement signal. Measurements in which a concentration series of cDNA were used to determine the lower detection limit (0.3 nM) and the sensitivity (7.2 mV / decade). Extracted and amplified target DNA from Mycobacterium tuberculosis-spiked human saliva-samples was also examined using the method. A clear differentiation between positive and negative material could be recognized with the help of the PAH / ssDNA-modified EIS sensor chips. All developed process steps were validated using fluorescence measurements as a reference method. With the PAH-modified capacitive field-effect biosensor, that was developed in this thesis, a quick, simple and inexpensive measurement platform for the DNA hybridization reaction is given. The detection of amplified genomic DNA from real Mycobacterium tuberculosis-spiked saliva samples underlines the potential of this procedure as a sensor approach for pathogen detection for medical applications

DNA sensing by electrocatalysis with hemoglobin

Electrocatalysis offers a means of electrochemical signal amplification, yet in DNA-based sensors, electrocatalysis has required high-density DNA films and strict assembly and passivation conditions. Here, we describe the use of hemoglobin as a robust and effective electron sink for electrocatalysis in DNA sensing on low-density DNA films. Protein shielding of the heme redox center minimizes direct reduction at the electrode surface and permits assays on low-density DNA films. Electrocatalysis with methylene blue that is covalently tethered to the DNA by a flexible alkyl chain linkage allows for efficient interactions with both the base stack and hemoglobin. Consistent suppression of the redox signal upon incorporation of a single cytosine-adenine (CA) mismatch in the DNA oligomer demonstrates that both the unamplified and the electrocatalytically amplified redox signals are generated through DNA-mediated charge transport. Electrocatalysis with hemoglobin is robust: It is stable to pH and temperature variations. The utility and applicability of electrocatalysis with hemoglobin is demonstrated through restriction enzyme detection, and an enhancement in sensitivity permits femtomole DNA sampling

Recent Progress in Optical Sensors for Biomedical Diagnostics

In recent years, several types of optical sensors have been probed for their aptitude in healthcare biosensing, making their applications in biomedical diagnostics a rapidly evolving subject. Optical sensors show versatility amongst different receptor types and even permit the integration of different detection mechanisms. Such conjugated sensing platforms facilitate the exploitation of their neoteric synergistic characteristics for sensor fabrication. This paper covers nearly 250 research articles since 2016 representing the emerging interest in rapid, reproducible and ultrasensitive assays in clinical analysis. Therefore, we present an elaborate review of biomedical diagnostics with the help of optical sensors working on varied principles such as surface plasmon resonance, localised surface plasmon resonance, evanescent wave fluorescence, bioluminescence and several others. These sensors are capable of investigating toxins, proteins, pathogens, disease biomarkers and whole cells in varied sensing media ranging from water to buffer to more complex environments such as serum, blood or urine. Hence, the recent trends discussed in this review hold enormous potential for the widespread use of optical sensors in early-stage disease prediction and point-of-care testing devices.DFG, 428780268, Biomimetische Rezeptoren auf NanoMIP-Basis zur Virenerkennung und -entfernung mittels integrierter Ansätz