In situ self-referenced intracellular two-electrode system for enhanced accuracy in single-cell analysis.

Since the emergence of single-cell electroanalysis, the two-electrode system has become the predominant electrochemical system for real-time behavioral analysis of single-cell and multicellular populations. However, due to the transmembrane placement of the two electrodes, cellular activities can be interrupted by the transmembrane potentials, and the test results are susceptible to influences from factors such as intracellular solution, membrane, and bulk solution. These limitations impede the advancement of single-cell analysis. Here, we propose a highly miniaturized and integrated in situ self-referenced intracellular two-electrode system (IS-SRITES), wherein both the working and reference electrodes are positioned inside the cell. Additionally, we demonstrated the stability (0.28 mV/h) of the solid-contact in situ Ag/AgCl reference electrode and the ability of the system to conduct standard electrochemical testing in a wide pH range (pH 6.0-8.0). Cell experiments confirmed the non-destructive performance of the electrode system towards cells and its capacity for real-time monitoring of intra- and extracellular pH values. Moreover, through equivalent circuits, finite element simulations, and drug delivery experiments, we illustrated that the IS-SRITES can yield more accurate test results and exhibit enhanced resistance to interference from the extracellular environment. Our proposed system holds the potential to enable the precise detection of intracellular substances and optimize the existing model of the electrode system for intracellular signal detection, thereby spearheading advancements in single-cell analysis.

Multifunctional IrOx Neural Probe for In Situ Dynamic Brain Hypoxia Evaluation.

Perioperative cerebral hypoxia and neonatal hypoxia-ischemic encephalopathy are the main triggers that lead to temporary or permanent brain dysfunction. The pathogenesis is intimately correlated to neural activities and the pH of the microenvironment, which calls for a high demand for in situ multitype physiological signal acquisition in the brain. However, conventional pH sensing neural interfaces cannot obtain the characteristics of multimodes, multichannels, and high spatial resolution of physiological signals simultaneously. Here, we report a multifunctional implantable iridium oxide (IrOx) neural probe (MIIONP) combined with electrophysiology recording, in situ pH sensing, and neural stimulation for real-time dynamic brain hypoxia evaluation. The neural probe modified with IrOx films exhibits outstanding electrophysiology recording and neural stimulation performance and long-term stable high spatial pH sensing resolution of about 100 µm, and the cytotoxicity of IrOx microelectrodes was investigated as well. In addition, 4 weeks' tracking of the same neuron firing and instantaneous population spike captured during electrical stimulation was achieved by MIIONP. Finally, in a mouse brain hypoxia model, the MIIONP has demonstrated the capability of synchronous in situ recording of the pH and neural firing changes in the brain, which has a valuable application in dynamic brain disease evaluation through real-time acquisition of multiple physiological signals.

A robust HD-sEMG sensor suitable for convenient acquisition of muscle activity in clinical post-stroke dysphagia.

Objective.A flexible high-density surface electromyography (HD-sEMG) sensor combined with an adaptive algorithm was used to collect and analyze the swallowing activities of patients with Post-stroke dysphagia.Approach.The electrode frame, modified electrode, and bonded substrate of the sensor were fabricated using a flexible printed circuit process, controlled drop coating, and molding, respectively. The adaptation algorithm was achieved by using Laplace and Teager-Kaiser energy operators to extract active segments, a cross-correlation coefficient matrix (CCCM) to evaluate synergy, and multi-frame real-time dynamic root mean square (RMS) to visualize spatiotemporal information to screen lesions and level of dysphagia. Finally, support vector machines (SVM) were adopted to explore the classification accuracy of sex, age, and lesion location with small sample sizes.Main results.The sensor not only has a basic low contact impedance (0.262 kΩ) and high signal-to-noise ratio (37.284 ± 1.088 dB) but also achieves other characteristics suitable for clinical applications, such as flexibility (747.67 kPa) and durability (1000 times) balance, simple operation (including initial, repeated, and replacement use), and low cost ($ 15.2). The three conclusions are as follows. CCCM can be used as a criterion for judging the unbalanced muscle region of the patient's neck and can accurately locate unbalanced muscles. The RMS cloud map provides the time consumption, swallowing times, and unbalanced areas. When the lesion location involves the left and right hemispheres simultaneously, it can be used as an evidence of relatively severely unbalanced areas. The classification accuracy of SVM in terms of sex, age, and lesion location was as high as 100%.Significance.The HD-sEMG sensor in this study and the adaptation algorithm will contribute to the establishment of a larger-scale database in the future to establish more detailed and accurate quantitative standards, which will be the basis for developing more optimized screening mechanisms and rehabilitation assessment methods.

A flexible neural implant with ultrathin substrate for low-invasive brain-computer interface applications.

Implantable brain-computer interface (BCI) devices are an effective tool to decipher fundamental brain mechanisms and treat neural diseases. However, traditional neural implants with rigid or bulky cross-sections cause trauma and decrease the quality of the neuronal signal. Here, we propose a MEMS-fabricated flexible interface device for BCI applications. The microdevice with a thin film substrate can be readily reduced to submicron scale for low-invasive implantation. An elaborate silicon shuttle with an improved structure is designed to reliably implant the flexible device into brain tissue. The flexible substrate is temporarily bonded to the silicon shuttle by polyethylene glycol. On the flexible substrate, eight electrodes with different diameters are distributed evenly for local field potential and neural spike recording, both of which are modified by Pt-black to enhance the charge storage capacity and reduce the impedance. The mechanical and electrochemical characteristics of this interface were investigated in vitro. In vivo, the small cross-section of the device promises reduced trauma, and the neuronal signals can still be recorded one month after implantation, demonstrating the promise of this kind of flexible BCI device as a low-invasive tool for brain-computer communication.

A novel flexible hydrogel electrode with a strong moisturizing ability for long-term EEG recording.

Objective. A novel flexible hydrogel electrode with a strong moisturizing ability was prepared for long-term electroencephalography (EEG) monitoring.Approach. The hydrogel was synthesized by polymerizing the N-acryloyl glycinamide monomer. And a proper amount of glycerin was added to the hydrogel to increase the moisture retention ability of the electrodes. The hydrogel shows high mechanical properties, and the liquid in the hydrogel produces a hydrating effect on the skin stratum corneum, which could decrease the contact impedance between skin and electrode. In addition, the installation of hydrogel electrode is very convenient, and the skin of the subject does not need to be abraded.Main results. Scanning electron microscope images show that there are a large number of micropores in the hydrogel, which provide storage space for water molecules. The average potential drift of the hydrogel electrode is relatively low (1.974 ± 0.560µV min-1). The average contact impedance of hydrogel electrode in forehead region and hair region are 6.43 ± 0.84 kΩ cm2and 13.15 ± 3.72 kΩ cm2, respectively. The result of open/closed paradigm, steady-state visual evoked potentials, and P300 visual evoked potential show that hydrogel electrode has excellent performance. Compared with the hydrogel without glycerin, the moisture retention ability of hydrogel containing glycerin was greatly improved.Significance.Compared with standard Ag/AgCl wet electrode, hydrogel electrode is more convenient to install and has strong moisture retention ability, which makes it have great potential in daily life for long-term EEG recording.

Dense Packed Drivable Optrode Array for Precise Optical Stimulation and Neural Recording in Multiple-Brain Regions.

The input-output function of neural networks is complicated due to the huge number of neurons and synapses, and some high-density implantable electrophysiology recording tools with a plane structure have been developed for neural circuit studies in recent years. However, traditional plane probes are limited by the record-only function and inability to monitor multiple-brain regions simultaneously, and the complete cognition of neural networks still has a long way away. Herein, we develop a three-dimensional (3D) high-density drivable optrode array for multiple-brain recording and precise optical stimulation simultaneously. The optrode array contains four-layer probes with 1024 microelectrodes and two thinned optical fibers assembled into a 3D-printed drivable module. The recording performance of microelectrodes is optimized by electrochemical modification, and precise implantation depth control of drivable optrodes is verified in agar. Moreover, in vivo experiments indicate neural activities from CA1 and dentate gyrus regions are monitored, and a tracking of the neuron firing for 2 weeks is achieved. The suppression of neuron firing by blue light has been realized through high-density optrodes during optogenetics experiments. With the feature of large-scale recording, optoelectronic integration, and 3D assembly, the high-density drivable optrode array possesses an important value in the research of brain diseases and neural networks.

Self-adaptive cardiac optogenetics device based on negative stretching-resistive strain sensor.

Precision medicine calls for high demand of continuous, closed-loop physiological monitoring and accurate control, especially for cardiovascular diseases. Cardiac optogenetics is promising for its superiority of cell selectivity and high time-space accuracy, but the efficacy of optogenetics relative to the input of light stimulus is detected and controlled separately by discrete instruments in vitro, which suffers from time retardation, energy consumption, and poor portability. Thus, a highly integrated system based on implantable sensors combining closed-loop self-monitoring with simultaneous treatment is highly desired. Here, we report a self-adaptive cardiac optogenetics system based on an original negative stretching-resistive strain sensor array for closed-loop heart rate recording and self-adaptive light intensity control. The strain sensor exhibits a dual and synchronous capability of precise monitor and physiological-electrical-optical regulation. In an in vivo ventricular tachycardia model, our system demonstrates the potential of a negative stretching-resistive device in controlling-in-sensor electronics for wearable/implantable autodiagnosis and telehealth applications.

Flexible Noncontact Sensing for Human-Machine Interaction.

From typical electrical appliances to thriving intelligent robots, the exchange of information between humans and machines has mainly relied on the contact sensor medium. However, this kind of contact interaction can cause severe problems, such as inevitable mechanical wear and cross-infection of bacteria or viruses between the users, especially during the COVID-19 pandemic. Therefore, revolutionary noncontact human-machine interaction (HMI) is highly desired in remote online detection and noncontact control systems. In this study, a flexible high-sensitivity humidity sensor and array are presented, fabricated by anchoring multilayer graphene (MG) into electrospun polyamide (PA) 66. The sensor works in noncontact mode for asthma detection, via monitoring the respiration rate in real time, and remote alarm systems and provides touchless interfaces in medicine delivery for bedridden patients. The physical structure of the large specific surface area and the chemical structure of the abundant water-absorbing functional groups of the PA66 nanofiber networks contribute to the high performance synergistically. This work can lead to a new era of noncontact HMI without the risk of contagiousness and provide a general and effective strategy for the development of smart electronics that require noncontact interaction.

Recombinase polymerase amplification with polymer flocculation sedimentation for rapid detection of Staphylococcus aureus in food samples.

Currently, rapid, sensitive, and convenient visual detection methods for Staphylococcus aureus (S. aureus) are scarce. In this study, a novel detection method based on recombinase polymerase amplification (RPA) and polymer flocculation sedimentation (PFS) was developed. Twelve effective primer combinations derived from four forward primers F1, F2, F3, F4, and three reverse primers R1, R2, R3 targeting the nuc gene of S. aureus were designed and screened by a polymerase chain reaction and RPA methods. RPA reaction conditions, including temperature, time, and volume as well as PEG8000 and NaCl concentrations range, were optimized. Moreover, the specificity and sensitivity of the RPA-PFS assay were further analyzed. Finally, the potential use of the RPA-PFS assay was evaluated using artificially S. aureus contaminated food samples, including pork, beef, shrimp, fish, cheese, cabbage, leftover rice, egg, milk, and orange juice. Results showed that the SA5 (F2/R2) combination was the optimal primer candidate. The optimal temperature range, the shortest time and the minimal volume of RPA reaction were 40-42 °C, 10 min and 10 µL, respectively and the optimal PEG8000/NaCl concentrations were 0.2 g/mL and 2.5 M, respectively, for the adsorption between magnetic beads and RPA products. The RPA-PFS method could detect as little as 13 fg genomic DNA of S. aureus and was also specific for five target S. aureus as well as twenty-seven non-target foodborne bacteria. The limit of detection of RPA-PFS for S. aureus in artificially contaminated food samples was 38 CFU/mL (g). Besides, RPA-PFS has directly been judged by the naked eye and has totally taken less than 20 min. In short, the assay RPA-PFS developed in this study is a rapid, sensitive, and specific visual detection method for S. aureus.

Biomarkers, oxidative stress and autophagy in skin aging.

Aging is a major cause of many degenerative diseases. The most intuitive consequence of aging is mainly manifested on the skin, resulting in cumulative changes in skin structure, function and appearance, such as increased wrinkles, laxity, elastosis, telangiectasia, and aberrant pigmentation of the skin. Unlike other organs of the human body, skin is not only inevitably affected by the intrinsic aging process, but also affected by various extrinsic environmental factors to accelerate aging, especially ultraviolet (UV) radiation. Skin aging is a highly complex and not fully understood process, and the lack of universal biomarkers for the definitive detection and evaluation of aging is also a major research challenge. Oxidative stress induced by the accumulation of reactive oxygen species (ROS) can lead to lipid, protein, nucleic acid and organelle damage, thus leading to the occurrence of cellular senescence, which is one of the core mechanisms mediating skin aging. Autophagy can maintain cellular homeostasis when faced with different stress conditions and is one of the survival mechanisms of cell resistance to intrinsic and extrinsic stress. Autophagy and aging have many features in common and may be associated with skin aging mediated by different factors. Here, we summarize the changes and biomarkers of skin aging, and discuss the effects of oxidative stress and autophagy on skin aging.

A Novel Bristle-Shaped Semi-Dry Electrode With Low Contact Impedance and Ease of Use Features for EEG Signal Measurements.

OBJECTIVE: In this paper, we present a novel soft bristle-shaped semi-dry electrode for electroencephalography (EEG) recording. Because the bristle-shaped structure with electric conductivity could overcome the obstacle of hair and enable direct connection to scalp, the semi-dry electrode could work with drinking water instead of saline water that was widely used in previous semi-dry or water electrodes to improve its convenience. The electrode consisted of conductive bristles and a 3D-printed casing. Carbon-coated nylon conductive bristles could achieve low impedance and soft properties of the semi-dry electrode. The bristles could spread on skin and realize a larger contact area. The carbon-coated conductive bristles could also continuously penetrate water into the corneum of skin to reduce contact impedance. The contact impedance of the bristle-shaped semi-dry electrode was similar to the traditional wet electrode, but much lower than dry electrode. Although the saline water had much lower impedance than drinking water, our electrode still achieved even lower skin-electrode contact impedance than previous semi-dry or water electrode with saline water. The alpha rhythms, P300 visual evoked potential, and steady-state visual evoked potential were, respectively, measured to evaluate the electrode performance for EEG recording.

Flexible bioelectrodes with enhanced wrinkle microstructures for reliable electrochemical modification and neuromodulation in vivo.

Limited electrode size with high electrochemical performance and reliability of modified materials are two of the main concerns for flexible neural electrodes in recent years. Here, an effective fabrication method of enhanced micro-scale wrinkles based on oil-pretreated hyperelastic substrates (PDMS and Ecoflex) is proposed for the application of microelectrode biosensors. Compared to pre-stretching or compressing methods, this approach has better advantages including compatibility with MEMS processes on wafer and easy replication. Wrinkled gold microelectrodes exhibit superior electrochemical properties than the flat one, and no crack or delamination occurs after electroplating PEDOT:PSS and platinum black on wrinkled microelectrodes. Cyclic voltammetry (CV) scanning for 2500 times is performed to investigate adhesion and stability of modified materials. For the modified microelectrodes, no significant change is observed in charge storage capacity (CSC) and impedance at 1 kHz, whereas PEDOT:PSS coated flat microelectrodes appears delamination. Ultrasonication and cycling forces are also conducted on modified microelectrodes, which demonstrates little influence on the wrinkled ones. Flexible wrinkled microelectrodes are further verified by in-vivo ECoG recordings combined with optogenetics in mice. These results highlight the importance of micro-structure in neural electrode design and tremendous application potentials in flexible electronics.

Sensitive and rapid visual detection of Salmonella Typhimurium in milk based on recombinase polymerase amplification with lateral flow dipsticks.

Salmonella Typhimurium (S. Typhimurium) can cause serious foodborne diseases. In this study, an assay combining recombinase polymerase amplification (RPA) with lateral flow dipsticks (LFD) was developed to detect S. Typhimurium in milk. The RPA forward primers STF1, STF2, STF3, the reverse primer STR labeled with digoxin, and the probe STProb labeled with FAM were designed and screened to produce RPA products for LFD detection. The RPA reaction volume, temperature, and time were then optimized, and the sensitivity and specificity of the developed method were analyzed. Finally, the RPA-LFD method was evaluated using milk artificially contaminated with S. Typhimurium. Results indicated that the primer pair STF1/STR is the optimal combination for detecting the bacterium. The minimum volume, shortest time, and optimal temperature of the RPA reaction were 10 µL, 10 min, and 40-42 °C, respectively. The limit of detection of RPA-LFD for detecting the genomic DNA of S. Typhimurium was 1 fg, which is 5 and 10 times lower than the corresponding limits of RPA-agarose gel electrophoresis (AGE) and PCR-AGE, respectively. Testing with 29 other foodborne bacteria as controls revealed that RPA-LFD was highly specific for S. Typhimurium. RPA-LFD can detect S. Typhimurium at concentrations as low as 1.95 CFU/mL in artificially inoculated milk samples and is thus 10 times more sensitive than PCR. Hence, the RPA-LFD assay established in this study could be a potential point-of-care/need test for S. Typhimurium, especially in areas with limited resources.

Development of duplex PCR-ELISA for simultaneous detection of Salmonella spp. and Escherichia coli O157: H7 in food.

In the current study, a duplex PCR-ELISA method was developed targeting the specific genes, invA of Salmonella spp. and rfbE of Escherichia coli O157: H7, to detect one or both bacteria in food. In brief, PCR product amplified by PCR primer labeled with digoxin at the 5'-end and a probe labeled with biotin at the 3'-end can form dimer by nucleic acid hybridization which can be captured by binding of biotin to streptomycin coated in ELISA plate before using enzyme-labeled anti-digoxin antibody and substrate to develop color. Also, evaluation of the duplex PCR-ELISA method was conducted in different food samples including milk, juice, cabbage, shrimp, chicken, pork and beef. Results indicated that the duplex PCR-ELISA developed here was specific when using 25 non-target bacteria strains as controls and was sensitive with a limit of detection (LOD) of 1 CFU/mL, 1, 000 times higher than that of the duplex PCR method and was repeatable regardless of inter- and intra-batch variations. The duplex PCR-ELISA method established in the present study has proven to be highly specific, sensitive and repeatable. It has the potential to be applied in such fields as clinical diagnosis of food-borne diseases, food hygiene monitoring and pathogen detection in food.