Label-free impedimetric immunosensor for point-of-care detection of COVID-19 antibodies.

The COVID-19 pandemic has posed enormous challenges for existing diagnostic tools to detect and monitor pathogens. Therefore, there is a need to develop point-of-care (POC) devices to perform fast, accurate, and accessible diagnostic methods to detect infections and monitor immune responses. Devices most amenable to miniaturization and suitable for POC applications are biosensors based on electrochemical detection. We have developed an impedimetric immunosensor based on an interdigitated microelectrode array (IMA) to detect and monitor SARS-CoV-2 antibodies in human serum. Conjugation chemistry was applied to functionalize and covalently immobilize the spike protein (S-protein) of SARS-CoV-2 on the surface of the IMA to serve as the recognition layer and specifically bind anti-spike antibodies. Antibodies bound to the S-proteins in the recognition layer result in an increase in capacitance and a consequent change in the impedance of the system. The impedimetric immunosensor is label-free and uses non-Faradaic impedance with low nonperturbing AC voltage for detection. The sensitivity of a capacitive immunosensor can be enhanced by simply tuning the ionic strength of the sample solution. The device exhibits an LOD of 0.4 BAU/ml, as determined from the standard curve using WHO IS for anti-SARS-CoV-2 immunoglobulins; this LOD is similar to the corresponding LODs reported for all validated and established commercial assays, which range from 0.41 to 4.81 BAU/ml. The proof-of-concept biosensor has been demonstrated to detect anti-spike antibodies in sera from patients infected with COVID-19 within 1 h. Photolithographically microfabricated interdigitated microelectrode array sensor chips & label-free impedimetric detection of COVID-19 antibody.

An impedimetric biosensor for COVID-19 serology test and modification of sensor performance via dielectrophoresis force.

Coronavirus disease 2019 (COVID-19) has caused significant global morbidity and mortality. The serology test that detects antibodies against the disease causative agent, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has often neglected value in supporting immunization policies and therapeutic decision-making. The ELISA-based antibody test is time-consuming and bulky. This work described a gold micro-interdigitated electrodes (IDE) biosensor for COVID antibody detection based on Electrochemical Impedance Spectroscopy (EIS) responses. The IDE architecture allows easy surface modification with the viral structure protein, Spike (S) protein, in the gap of the electrode digits to selectively capture anti-S antibodies in buffer solutions or human sera. Two strategies were employed to resolve the low sensitivity issue of non-faradic impedimetric sensors and the sensor fouling phenomenon when using the serum. One uses secondary antibody-gold nanoparticle (AuNP) conjugates to further distinguish anti-S antibodies from the non-specific binding and obtain a more significant impedance change. The second strategy consists of increasing the concentration of target antibodies in the gap of IDEs by inducing an AC electrokinetic effect such as dielectrophoresis (DEP). AuNP and DEP methods reached a limit of detection of 200 ng/mL and 2 µg/mL, respectively using purified antibodies in buffer, while the DEP method achieved a faster testing time of only 30 min. Both strategies could qualitatively distinguish COVID-19 antibody-positive and -negative sera. Our work, especially the impedimetric detection of COVID-19 antibodies under the assistance of the DEP force presents a promising path toward rapid, point-of-care solutions for COVID-19 serology tests.

Ultrasound-assisted magnetic nanoparticle-based gene delivery.

Targeted gene delivery is important in biomedical research and applications. In this paper, we synergistically combine non-viral chemical materials, magnetic nanoparticles (MNPs), and a physical technique, low-intensity pulsed ultrasound (LIPUS), to achieve efficient and targeted gene delivery. The MNPs are iron oxide super-paramagnetic nanoparticles, coated with polyethyleneimine (PEI), which makes a high positive surface charge and is favorable for the binding of genetic materials. Due to the paramagnetic properties of the MNPs, the application of an external magnetic field increases transfection efficiency while LIPUS stimulation enhances cell viability and permeability. We found that stimulation at the intensity of 30 mW/cm2 for 10 minutes yields optimal results with a minimal adverse effect on the cells. By combining the effect of the external magnetic field and LIPUS, the genetic material (GFP or Cherry Red plasmid) can enter the cells. The flow cytometry results showed that by using just a magnetic field to direct the genetic material, the transfection efficiency on HEK 293 cells that were treated by our MNPs was 56.1%. Coupled with LIPUS stimulation, it increased to 61.5% or 19% higher than the positive control (Lipofectamine 2000). Besides, compared with the positive control, our method showed less toxicity. Cell viability after transfection was 63.61%, which is 19% higher than the standard transfection technique. In conclusion, we designed a new gene-delivery method that is affordable, targeted, shows low-toxicity, yet high transfection efficiency, compared to other conventional approaches.

Single ascospore detection for the forecasting of Sclerotinia stem rot of canola.

Smart-agriculture technologies comprise a set of management systems designed to sustainably increase the efficiency and productivity of farming. In this paper, we present a lab-on-a-chip device that can be employed as a plant disease forecasting tool for canola crop. Our device can be employed as a platform to forecast potential outbreaks of one of the most devastating diseases of canola and other crops, Sclerotinia stem rot. The system consists of a microfluidic chip capable of detecting single airborne Sclerotinia sclerotiorum ascospores. Target ascospores are injected into the chip and selectively captured by dielectrophoresis, while other spores in the sample are flushed away. Afterward, captured ascospores are released into the flow stream of the channel and are detected employing electrochemical impedance spectroscopy and coplanar microelectrodes. Our device provides a design for a low-cost, miniaturized, and automated platform technology for airborne spore detection and disease prevention.

DNA aptamer-based non-faradaic impedance biosensor for detecting E. coli.

Developing a real-time, portable, and inexpensive sensor for pathogenic bacteria is crucial since the conventional detection approaches such as enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) assays are high cost, time-consuming, and require an expert operator. Here we present a portable, inexpensive, and convenient impedance-based biosensor using Interdigitated Electrode (IDE) arrays to detect Escherichia coli (E. coli) as a model to demonstrate the feasibility of an impedance-based biosensor. We manipulated the affinity of the IDE array towards E. coli (E. coli BL21 series) by functionalizing the IDEs' surface with an E. coli outer membrane protein (OMP) Ag1 Aptamer. To determine the dominant factors affecting the sensitivity and the performance of the biosensor in detecting E. coli, we investigated the roles of the substrate material used in the fabrication of the IDE, the concentration of the aptamer, and the composition of the carboxy aliphatic thiol mixture used in the pre-treatment of the IDE surface. In the sensing experiments we used an E. coli concentration range of 25-1000 cfu mL-1 and confirmed the binding of the OMP Ag1 Aptamer to the outer membrane protein of the E. coli by Field Emission Scanning Electron Microscopy (FESEM), Optical Microscopy, and Atomic Force Microscopy (AFM). By tuning the surface chemistry, the IDEs' substrate material, and the concentration of the OMP Ag1 Aptamer, our sensor could detect E. coli with the analytical sensitivity of approximately 1.8 Ohm/cfu and limit of detection of 9 cfu mL-1. We found that the molecular composition of the self-assembled monolayer (SAM) formed on the top of the IDEs before the attachment of the OMP Ag1 Aptamer significantly impacted the sensitivity of the sensor. Notably, with straightforward changes to the molecular recognition elements, this platform device can be used to detect a wide range of other microorganisms and chemicals relevant for environmental monitoring and public health.

Non-invasive Point-of-Care Device To Diagnose Acute Mesenteric Ischemia.

Inadequate blood supply to the intestine can lead to acute mesenteric ischemia (AMI), with a mortality rate ranging from 60% to 90%. This high mortality rate is partially due to late detection and the lack of efficient early diagnostic tests. There is an urgent need for a point-of-care tool for immediate bedside diagnosis. Here we present for the first time a rapid and non-invasive electrochemical biosensor device based on non-faradic impedance spectroscopy to detect intestinal fatty-acid binding protein (I-FABP) as an indication of AMI. The electrochemical biosensors consist of gold interdigitated electrodes that were fabricated using photolithographic techniques on top of silicon dioxide substrates. The electrode surfaces were functionalized with an I-FABP capture antibody (CAnB) to entice the target protein, while gold nanoparticles (GNPs) functionalized with detection antibodies (DAnB-GNPs) were utilized as a novel mechanism to enhance the detection signal. Quantification of the I-FABP concentration in the medium depended on its attachment to CAnB and DAnB-GNPs in a sandwich manner, where the latter boosts the impedance signal through its binding to the I-FABP. This non-invasive non-faradic electric biosensor device demonstrates the potential for bench-to-bedside translation with the goal of decreasing morbidity and mortality from AMI.

Immuno-impedimetric Biosensor for Onsite Monitoring of Ascospores and Forecasting of Sclerotinia Stem Rot of Canola.

Sclerotinia stem rot, caused by the fungal pathogen Sclerotinia sclerotiorum, is a destructive disease of canola and many other broadleaf crops. The primary inoculum responsible for initiating Sclerotinia epidemics is airborne ascospores released from the apothecia of sclerotia. Timely detection of the presence of airborne ascospores can serve as an early-warning system for forecasting and management of the disease. A major challenge is to develop a portable and automated device which can be deployed onsite to detect and quantify the presence of minute quantities of ascospores in the air and serves as a unit in a network of systems for forecasting of the epidemic. In this communication, we present the development of an impedimetric non-Faradaic biosensor based on anti-S. sclerotiorum polyclonal antibodies as probes to selectively capture the ascospores and sense their binding by an impedance based interdigitated electrode which was found to directly and unambiguously correlate the number of ascospores on sensor surface with the impedance response.

Transfection of Difficult-to-Transfect Rat Primary Cortical Neurons with Magnetic Nanoparticles.

The efficient cell transfection method is vital for various biomedical applications, such as the CRISPR-Cas9 technique. Current cell transfection methods, including lipofectamine, calcium phosphate co-precipitation, nucleofection, and viral infection are not equally efficient for various cells and have their disadvantages. In this study, a magnetic nanoparticle (MNP)-based method was introduced for delivering both FITC dye and a functional EGFP gene into easy-to-transfect HEK cells and difficult-to-transfect rat primary cortical neurons. The transfection efficacy could be controlled in both time-dependent and magnetic strength-dependent manner. This cell transfection method could have substantial potential for targeted drug delivery.

Using Impedance Measurements to Characterize Surface Modified with Gold Nanoparticles.

With the increased practice of preventative healthcare to help reduce costs worldwide, sensor technology improvement is vital to patient care. Point-of-care (POC) diagnostics can reduce time and lower labor in testing, and can effectively avoid transporting costs because of portable designs. Label-free detection allows for greater versatility in the detection of biological molecules. Here, we describe the use of an impedance-based POC biosensor that can detect changes in the surface modification of a micro-fabricated chip using impedance spectroscopy. Gold nanoparticles (GNPs) have been employed to evaluate the sensing ability of our new chip using impedance measurements. Furthermore, we used impedance measurements to monitor surface functionalization progress on the sensor's interdigitated electrodes (IDEs). Electrodes made from aluminum and gold were employed and the results were analyzed to compare the impact of electrode material. GNPs coated with mercaptoundecanoic acid were also used as a model of biomolecules to greatly enhance chemical affinity to the silicon substrate. The portable sensor can be used as an alternative technology to ELISA (enzyme-linked immunosorbent assays) and polymerase chain reaction (PCR)-based techniques. This system has advantages over PCR and ELISA both in the amount of time required for testing and the ease of use of our sensor. With other techniques, larger, expensive equipment must be utilized in a lab environment, and procedures have to be carried out by trained professionals. The simplicity of our sensor system can lead to an automated and portable sensing system.

Gold nanoparticle-filled biodegradable photopolymer scaffolds induced muscle remodeling: in vitro and in vivo findings.

Therapeutic stem cell transplantation bears the promise of new directions in organ and tissue replacement, but a number of its difficulties and perils are also well known. Our goal was to develop a method of transplantation by which the transplanted cells remain confined to the transplantation site and induce favorable processes. With the help of mask-projection excimer laser stereolithography, 3D hybrid nanoscaffolds were fabricated from biodegradable, photocurable PPF:DEF resin with incorporated gold nanoparticles (Au NPs). The scaffolds were tested in vitro and in vivo in order to find out about their biocompatibility and fitness for our purposes. In vitro, macrophages and mouse autologous adipose stem cells (ASCs) were seeded over the hybrid scaffolds and non-hybrid (with Au NPs) scaffolds for 4days. The hybrid nanocomposite greater stem cell dispension and stem cell adhesion than PPF scaffolds without Au NPs, but such a difference was not seen in the case of macrophages. In vivo, stem cells, scaffoldings and scaffoldings covered in stem cells were transplanted under the back skin of mice. After 14days, blood samples were taken and the affected skin area was excised. Cytokine and chemokine profiling did not indicate elevated immunomediators in the sera of experimental animals. Interestingly, the autologous-stem-cell-seeded hybrid nanocomposite scaffold induced muscle tissue regeneration after experimental wound generation in vivo. We could not observe such stem cell-induced tissue regeneration when no scaffolding was used. We conclude that PPF:DEF resin nanoscaffolds with incorporated gold nanoparticles offer a safe and efficient alternative for the enhancement of local tissue remodeling. The results also support the idea that adipose derived stem cells are an optimal cell type for the purposes of regenerative musculoskeletal tissue engineering.

PEGylated gold nanorods as optical trackers for biomedical applications: an in vivo and in vitro comparative study.

Gold nanorods (AuNRs) are eligible for a variety of biological applications including cell imaging, sensing, and photothermal therapy thanks to their optical properties. The aim of this work is to show how AuNRs could be employed as non-photobleachable optical contrast agents for biomedical applications. In order to demonstrate the feasibility of their use as optical trackers, we employed two-photon emission confocal microscopy on cells incubated with PEGylated AuNRs. Remarkably, AuNRs were localized mostly in the perinuclear zone and microscopy characterization showed the presence of a considerable number of rods inside cell nuclei. Furthermore, we estimated the toxicity and the efficiency of cellular uptake of the PEGylated AuNRs as a function of administered dose on HeLa/3T3 cell lines and on zebrafish during development, employed as an in vivo model. Eventually, we observed good agreement between in vivo and in vitro experiments. The employed AuNRs were prepared through a photochemical protocol here improved by tuning the amount of the cationic surfactant cetyltrimethylammonium bromide for the achievement of AuNRs at two different aspect ratios. Furthermore we also investigated if the AuNR aspect ratio influenced the toxicity and the efficiency of cellular uptake of the PEGylated AuNRs in HeLa/3T3 cell lines and in zebrafish embryos.

Effect of Anderson localization on light emission from gold nanoparticle aggregates.

The localization of light known as Anderson localization is a common phenomenon characterizing aggregates of metallic nanostructures. The electromagnetic energy of visible light can be localized inside nanostructures below the diffraction limit by converting the optical modes into nonradiative surface plasmon resonances. The energy of the confined photons is correlated to the size and shape of the nanostructured system. In this work, we studied the photoluminescence dependence of aggregates of 14 nm diameter gold nanoparticles (AuNPs) synthesized by drop-casting a liquid suspension on two different substrates of glass and quartz. The AuNP aggregates were characterized by electron microscopy, X-ray diffraction and X-ray photoelectron spectroscopy. The dielectric constant of the surrounding medium plays a crucial role in determining the aggregate geometry, which affects the Anderson localization of light in the aggregates and hence causes a red-shift in the plasmonic resonance and in the photoluminescence emission. The geometry of the gold nanoparticle aggregates determine the strength of the Anderson localization, and hence, the light emission from the aggregates. The photoluminescence lifetime was found to be dependent on the AuNP aggregate geometry and the dielectric constant of the medium.

Nanocomposite scaffold fabrication by incorporating gold nanoparticles into biodegradable polymer matrix: Synthesis, characterization, and photothermal effect.

Nanoparticle incorporation into scaffold materials is a valuable route to deliver various therapeutic agents, such as drug molecules or large biomolecules, proteins (e.g. DNA or RNA) into their targets. In particular, gold nanoparticles (Au NPs) with their low inherent toxicity, tunable stability and high surface area provide unique attributes facilitating new delivery strategies. A biodegradable, photocurable polymer resin, polypropylene fumarate (PPF) along with Au NPs were utilized to synthesize a hybrid nanocomposite resin, directly exploitable in stereolithography (SL) processes. To increase the particles' colloidal stability, the Au NP nanofillers were coated with polyvinyl pyrrolidone (PVP). The resulting resin was used to fabricate a new type of composite scaffold via mask projection excimer laser stereolithography. The thermal properties of the nanocomposite scaffolds were found to be sensitive to the concentration of NPs. The mechanical properties were augmented by the NPs up to 0.16µM, though further increase in the concentration led to a gradual decrease. Au NP incorporation rendered the biopolymer scaffolds photosensitive, i.e. the presence of Au NPs enhanced the optical absorption of the scaffolds as well, leading to possible localized temperature rise when irradiated with 532nm laser, known as the photothermal effect.