Ultrasensitive Electrochemical Immunosensors Using Nanobodies as Biocompatible Sniffer Tools of Agricultural Contaminants and Human Disease Biomarkers.

Nanobodies (Nbs) are known as camelid single-domain fragments or variable heavy chain antibodies (VHH) that in vitro recognize the antigens (Ag) similar to full-size antibodies (Abs) and in vivo allow immunoreactions with biomolecule cavities inaccessible to conventional Abs. Currently, Nbs are widely used for clinical treatments due to their remarkably improved performance, ease of production, thermal robustness, superior physical and chemical properties. Interestingly, Nbs are also very promising bioreceptors for future rapid and portable immunoassays, compared to those using unstable full-size antibodies. For all these reasons, Nbs are excellent candidates in ecological risk assessments and advanced medicine, enabling the development of ultrasensitive biosensing platforms. In this review, immobilization strategies of Nbs on conductive supports for enhanced electrochemical immune detection of food contaminants (Fcont) and human biomarkers (Hbio) are discussed. In the case of Fcont, the direct competitive immunoassay detection using coating antigen solid surface is the most commonly used approach for efficient Nbs capture which was characterized with cyclic voltammetry (CV) and differential pulse voltammetry (DPV) when the signal decays for increasing concentrations of free antigen prepared in aqueous solutions. In contrast, for the Hbio investigations on thiolated gold electrodes, increases in amperometric and electrochemical impedance spectroscopy (EIS) signals were recorded, with increases in the antigen concentrations prepared in PBS or spiked real human samples.

Updates on the Biofunctionalization of Gold Nanoparticles for the Rapid and Sensitive Multiplatform Diagnosis of SARS-CoV-2 Virus and Its Proteins: From Computational Models to Validation in Human Samples.

Since the outbreak of the pandemic respiratory virus SARS-CoV-2 (COVID-19), academic communities and governments/private companies have used several detection techniques based on gold nanoparticles (AuNPs). In this emergency context, colloidal AuNPs are highly valuable easy-to-synthesize biocompatible materials that can be used for different functionalization strategies and rapid viral immunodiagnosis. In this review, the latest multidisciplinary developments in the bioconjugation of AuNPs for the detection of SARS-CoV-2 virus and its proteins in (spiked) real samples are discussed for the first time, with reference to the optimal parameters provided by three approaches: one theoretical, via computational prediction, and two experimental, using dry and wet chemistry based on single/multistep protocols. Overall, to achieve high specificity and low detection limits for the target viral biomolecules, optimal running buffers for bioreagent dilutions and nanostructure washes should be validated before conducting optical, electrochemical, and acoustic biosensing investigations. Indeed, there is plenty of room for improvement in using gold nanomaterials as stable platforms for ultrasensitive and simultaneous "in vitro" detection by the untrained public of the whole SARS-CoV-2 virus, its proteins, and specific developed IgA/IgM/IgG antibodies (Ab) in bodily fluids. Hence, the lateral flow assay (LFA) approach is a quick and judicious solution to combating the pandemic. In this context, the author classifies LFAs according to four generations to guide readers in the future development of multifunctional biosensing platforms. Undoubtedly, the LFA kit market will continue to improve, adapting researchers' multidetection platforms for smartphones with easy-to-analyze results, and establishing user-friendly tools for more effective preventive and medical treatments.

Use of Cysteamine and Glutaraldehyde Chemicals for Robust Functionalization of Substrates with Protein Biomarkers-An Overview on the Construction of Biosensors with Different Transductions.

Currently, several biosensors are reported to confirm the absence/presence of an abnormal level of specific human biomarkers in research laboratories. Unfortunately, public marketing and/or pharmacy accessibility are not yet possible for many bodily fluid biomarkers. The questions are numerous, starting from the preparation of the substrates, the wet/dry form of recognizing the (bio)ligands, the exposure time, and the choice of the running buffers. In this context, for the first time, the present overview summarizes the pre-functionalization of standard and nanostructured solid/flexible supports with cysteamine (Cys) and glutaraldehyde (GA) chemicals for robust protein immobilization and detection of biomarkers in body fluids (serum, saliva, and urine) using three transductions: piezoelectrical, electrochemical, and optical, respectively. Thus, the reader can easily access and compare step-by-step conjugate protocols published over the past 10 years. In conclusion, Cys/GA chemistry seems widely used for electrochemical sensing applications with different types of recorded signals, either current, potential, or impedance. On the other hand, piezoelectric detection via quartz crystal microbalance (QCM) and optical detection by surface plasmon resonance (LSPR)/surface-enhanced Raman spectroscopy (SERS) are ultrasensitive platforms and very good candidates for the miniaturization of medical devices in the near future.

Chemosensing on Miniaturized Plasmonic Substrates.

Round, small-sized coverslips were coated for the first time with thin layers of indium tin oxide (ITO, 10-40 nm)/gold (Au, 2-8 nm) and annealed at 550 °C for several hours. The resulting nanostructures on miniaturized substrates were further optimized for the localized surface plasmon resonance (LSPR) chemosensing of a model molecule-1,2-bis-(4-ppyridyl)-ethene (BPE)-with a detection limit of 10-12 M BPE in an aqueous solution. All the fabrication steps of plasmonic-annealed platforms were characterized using scanning electron microscopy (SEM) and atomic force microscopy (AFM).

Influence of Saline Buffers over the Stability of High-Annealed Gold Nanoparticles Formed on Coverslips for Biological and Chemosensing Applications.

Herein, coverslips were used as solid supports for the synthesis of gold nanoparticles (AuNPs) in three steps: (i) detergent cleaning, (ii) evaporation of 4 nm gold film and (iii) exposure at high annealing temperature (550 °C) for 3 h. Such active gold nanostructured supports were investigated for their stability performances in aqueous saline buffers for new assessments of chemical sensing. Two model buffers, namely saline-sodium phosphate-EDTA buffer (SSPE) and phosphate buffer saline (PBS), that are often used in the construction of (bio)sensors, are selected for the optical and microscopic investigations of their influence over the stability of annealed AuNPs on coverslips when using a dropping procedure under dry and wet media working conditions. A study over five weeks monitoring the evolution of the localized surface plasmon resonance (LSPR) chemosensing of 1,2-bis-(4-pyridyl)-ethene (BPE) is discussed. It is concluded that the optimal sensing configuration is based on annealed AuNPs exposed to saline buffers under wet media conditions (overnight at 4 °C) and functionalized with BPE concentrations (10-3-10-11 M) with the highest LSPR spectra after two weeks.

Acoustic Multi-Detection of Gliadin Using QCM Crystals Patterned with Controlled Sectors of TEM Grid and Annealed Nanoislands on Gold Electrode.

Celiac diseases are a group of gluten ingestion-correlated pathologies that are widespread and, in some cases, very dangerous for human health. The only effective treatment is the elimination of gluten from the diet throughout life. Nowadays, the food industries are very interested in cheap, easy-to-handle methods for detecting gluten in food, in order to provide their consumers with safe and high-quality food. Here, for the first time, the manufacture of controlled micropatterns of annealed gold nanoislands (AuNIs) on a single QCM crystal (QCM-color) and their biofunctionalization for the specific detection of traces of gliadin is reported. In addition, the modified quartz crystal with a TEM grid and 30 nm Au (Q-TEM grid crystal) is proposed as an acoustic sensitive biosensing platform for the rapid screening of the gliadin content in real food products.

Correction: Facile, wafer-scale compatible growth of ZnO nanowires via chemical bath deposition: assessment of zinc ion contribution and other limiting factors.

[This corrects the article DOI: 10.1039/D0NA00434K.].

Facile, wafer-scale compatible growth of ZnO nanowires via chemical bath deposition: assessment of zinc ion contribution and other limiting factors.

ZnO is a highly promising, multifunctional nanomaterial having various versatile applications in the fields of sensors, optoelectronics, photovoltaics, photocatalysts and water purification. However, the real challenge lies in producing large scale, well-aligned, highly reproducible ZnO nanowires (NWs) using low cost techniques. This large-scale production of ZnO NWs has stunted the development and practical usage of these NWs in fast rising fields such as photocatalysis or in photovoltaic applications. The present article shows an effective, simple approach for the uniform, aligned growth of ZnO NWs on entire silicon wafers (sizes 3 or 4 inches), using a low-temperature Chemical Bath Deposition (CBD) technique. In addition to this, a systematic study of the substrate size dependent growth of NWs has been conducted to better understand the effect of the limitation in the deposition rate of Zn2+ ions on the growth of NWs. The growth rate of ZnO NWs is seen to have a strong relationship with the substrate size. Also, the loading efficiency of the Zn2+ ions is higher in ZnO NWs grown on a 3-inch silicon wafer in comparison to those grown on a small piece. An in-depth time dependent growth study conducted on entire 3-inch wafers to track the morphological evolution (length, diameter and number of the NWs) reveals that the growth rate of the length of the NWs reaches a saturation state in a short time span of 20 min. Assessment of the overall homogeneity of the NWs grown on the 3-inch wafer and simultaneous growth on two entire 4-inch silicon wafers has also been demonstrated in this article. This demonstration of large-scale, well-aligned controllable, aligned growth of ZnO NWs on entire silicon wafers is a first step towards NW based devices especially for applications such as photovoltaic, water purification, photocatalysis or biomedical applications.

Robust SERS Platforms Based on Annealed Gold Nanostructures Formed on Ultrafine Glass Substrates for Various (Bio)Applications.

In this study, stable gold nanoparticles (AuNPs) are fabricated for the first time on commercial ultrafine glass coverslips coated with gold thin layers (2 nm, 4 nm, 6 nm, and 8 nm) at 25 °C and annealed at high temperatures (350 °C, 450 °C, and 550 °C) on a hot plate for different periods of time. Such gold nanostructured coverslips were systematically tested via surface enhanced Raman spectroscopy (SERS) to identify their spectral performances in the presence of different concentrations of a model molecule, namely 1,2-bis-(4-pyridyl)-ethene (BPE). By using these SERS platforms, it is possible to detect BPE traces (10-12 M) in aqueous solutions in 120 s. The stability of SERS spectra over five weeks of thiol-DNA probe (2 µL) deposited on gold nano-structured coverslip is also reported.

Microwave Spectroscopic Detection of Human Hsp70 Protein on Annealed Gold Nanostructures on ITO Glass Strips.

Conductive indium-tin oxide (ITO) and non-conductive glass substrates were successfully modified with embedded gold nanoparticles (AuNPs) formed by controlled thermal annealing at 550 °C for 8 h in a preselected oven. The authors characterized the formation of AuNPs using two microscopic techniques: scanning electron microscopy (SEM) and atomic force microscopy (AFM). The analytical performances of the nanostructured-glasses were compared regarding biosensing of Hsp70, an ATP-driven molecular chaperone. In this work, the human heat-shock protein (Hsp70), was chosen as a model biomarker of body stress disorders for microwave spectroscopic investigations. It was found that microwave screening at 4 GHz allowed for the first time the detection of 12 ng/µL/cm² of Hsp70.

Fabrication of Annealed Gold Nanostructures on Pre-Treated Glow-Discharge Cleaned Glasses and Their Used for Localized Surface Plasmon Resonance (LSPR) and Surface Enhanced Raman Spectroscopy (SERS) Detection of Adsorbed (Bio)molecules.

Metallic nanoparticles are considered as active supports in the development of specific chemical or biological biosensors. Well-organized nanoparticles can be prepared either through expensive (e.g., electron beam lithography) or inexpensive (e.g., thermal synthesis) approaches where different shapes of nanoparticles are easily obtained over large solid surfaces. Herein, the authors propose a low-cost thermal synthesis of active plasmonic nanostructures on thin gold layers modified glass supports after 1 h holding on a hot plate (~350 °C). The resulted annealed nanoparticles proved a good reproducibility of localized surface plasmon resonance (LSPR) and surface enhanced Raman spectroscopy (SERS) optical responses and where used for the detection of low concentrations of two model (bio)chemical molecules, namely the human cytochrome b5 (Cyt-b5) and trans-1,2-bis(4-pyridyl)ethylene (BPE).

Fate and Characterization Factors of Nanoparticles in Seventeen Subcontinental Freshwaters: A Case Study on Copper Nanoparticles.

The lack of characterization factors (CFs) for engineered nanoparticles (ENPs) hampers the application of life cycle assessment (LCA) methodology in evaluating the potential environmental impacts of nanomaterials. Here, the framework of the USEtox model has been selected to solve this problem. On the basis of colloid science, a fate model for ENPs has been developed to calculate the freshwater fate factor (FF) of ENPs. We also give the recommendations for using the hydrological data from the USEtox model. The functionality of our fate model is proved by comparing our computed results with the reported scenarios in North America, Switzerland, and Europe. As a case study, a literature survey of the nano-Cu toxicology values has been performed to calculate the effect factor (EF). Seventeen freshwater CFs of nano-Cu are proposed as recommended values for subcontinental regions. Depending on the regions and the properties of the ENPs, the region most likely to be affected by nano-Cu is Africa (CF of 11.11 × 10(3) CTUe, comparative toxic units) and the least likely is north Australia (CF of 3.87 × 10(3) CTUe). Furthermore, from the sensitivity analysis of the fate model, 13 input parameters (such as depth of freshwater, radius of ENPs) show vastly different degrees of influence on the outcomes. The characterization of suspended particles in freshwater and the dissolution rate of ENPs are two significant factors.

Measurement of Bacterial Bioluminescence Intensity and Spectrum: Current Physical Techniques and Principles.

: Bioluminescence is light production by living organisms, which can be observed in numerous marine creatures and some terrestrial invertebrates. More specifically, bacterial bioluminescence is the "cold light" produced and emitted by bacterial cells, including both wild-type luminescent and genetically engineered bacteria. Because of the lively interplay of synthetic biology, microbiology, toxicology, and biophysics, different configurations of whole-cell biosensors based on bacterial bioluminescence have been designed and are widely used in different fields, such as ecotoxicology, food toxicity, and environmental pollution. This chapter first discusses the background of the bioluminescence phenomenon in terms of optical spectrum. Platforms for bacterial bioluminescence detection using various techniques are then introduced, such as a photomultiplier tube, charge-coupled device (CCD) camera, micro-electro-mechanical systems (MEMS), and complementary metal-oxide-semiconductor (CMOS) based integrated circuit. Furthermore, some typical biochemical methods to optimize the analytical performances of bacterial bioluminescent biosensors/assays are reviewed, followed by a presentation of author's recent work concerning the improved sensitivity of a bioluminescent assay for pesticides. Finally, bacterial bioluminescence as implemented in eukaryotic cells, bioluminescent imaging, and cancer cell therapies is discussed.

A facile and cost-effective TEM grid approach to design gold nano-structured substrates for high throughput plasmonic sensitive detection of biomolecules.

A commercial TEM grid was used as a mask for the creation of extremely well-organized gold micro-/nano-structures on a glass substrate via a high temperature annealing process at 500 °C. The structured substrate was (bio)functionalized and used for the high throughput LSPR immunosensing of different concentrations of a model protein named bovine serum albumin.

Fabrication of an atrazine acoustic immunosensor based on a drop-deposition procedure.

Among the various novel analytical systems, immunosensors based on acoustic waves are of emerging interest because of their good sensitivity, real-time monitoring capability, and experimental simplicity. In this work, piezoelectric immunosensors were constructed for the detection of atrazine through the immobilization of specific monoclonal anti-atrazine antibodies on thiolated modified quartz crystal microbalances (QCMs). The immunoassay was conducted by a novel drop-deposition procedure using different atrazine dilutions in phosphate buffer solution ranging from 10(-10) to 10(-1) mg/mL. The immunoreactions between varying contents of atrazine and its antibody were dynamically exhibited through in situ monitoring of the frequency and motional resistance changes over 20 min. Thus, atrazine recognition by the anti-atrazine antibody leads to a decrease of the resonant frequency that is proportional to a given atrazine concentration. Interestingly, the motional resistance also increased proportionally during the measurements, which could be attributed to the specific viscoelastic properties and/or conformation changes of the antibodies once the immunoreactions occurred. By combining the measurements of frequency with those of motional resistance, additional information was provided about the interaction between the atrazine-named antigen and its respective antibody. Finally, the analytical specificity of the immunosensor to atrazine was evaluated through the response to a nonspecific anti-human IgG antibody-modified QCM crystal under the same drop conditions.

EIS microfluidic chips for flow immunoassay and ultrasensitive cholera toxin detection.

A flow-injection impedimetric immunosensor for the sensitive, direct and label-free detection of cholera toxin is reported. A limit of detection smaller than 10 pM was achieved, a value thousands of times lower than the lethal dose. The developed chips fulfil the requirement of low cost and quick reply of the assay and are expected to enable field screening, prompt diagnosis and medical intervention without the need of specialized personnel and expensive equipment, a perspective of special relevance for use in developing countries. Since the chip layout includes two sensing areas each one with a 2 × 2 sensor array, our biochips can allow statistical or (alternatively) multiplex analysis of biorecognition events between antibodies immobilized on each working electrode and different antigens flowing into the chamber.

Real-time monitoring of copper ions-induced cytotoxicity by EIS cell chips.

An important goal of biomedical research is the development of tools for high-throughput evaluation of drug effects and cytotoxicity tests. Here we demonstrate EIS cell chips able to monitor cell growth, morphology, adhesion and their changes as a consequence of treatment with drugs or toxic compounds. As a case study, we investigate the uptake of copper ions and its effect on two cell lines: B104 and HeLa cells. For further understanding, we also carried out in parallel with EIS studies, a complete characterization of cell morphology and changes induced by copper ions through complementary methodologies (including state-of-the-art AFM, viability test and Western blot). Our results reveal a strong correlation between EIS data and both MTT test and AFM characterization so our chip can be used as powerful tools in all biology lab in combination with other standard methods giving additional information that can be useful in a complete and deep investigation of a biological process. This chip can be used even alone replacing in vitro drug tests based on conventional biochemical methods, being very cheap and reusable and allowing to perform cytotoxicity tests without using any expensive reagent or equipment.