Micromachined Sensors for Single-Cell Signalling

The fabrication of microelectrodes integrated within microtitre chambers for ultra-low volume amperometric determination of lactate continues to be of interest in the subject of cell screening. Devices were fabricated using photolithography to give highly reproducible sensors integrated within high aspect ratio analytical chambers with volumes of between 160 ~ 400 pL. These chambers were either circular or square with a depth of 20 ?m, defined using the photoepoxy, SU8. In addition to the 3- microelectrode sensor, a 2-microelectrode sensor was also studied, which consists of a working microelectrode and an internal pseudo-microreference (combined with the counter electrode). The electrochemical characterization of a miniaturized sensor using a model redox compound of FMCA was discussed. Meanwhile, the detection of hydrogen peroxide, the redox enzyme linked electrochemical assay of glucose and the regeneration of the micro planar electrodes were investigated. 'Bulk' and pL-scale lactate measurements were carried out using the micromachined sensors. A microfluidic dispensation system was developed to deliver very low titres (6.5 pL) into a low volume micro-electrochemical cell. The determination of lactate was optimised using an enzyme-linked assay based upon lactate oxidase. This involved the amperometric determination of hydrogen peroxide at 640 mV vs. an internal pseudo AglAgCl reference (in the 2 electrode configuration). The system (including the microfluidic device, the microfluidic dispenser) had an observed detection limit of 15-fmol of lactate (as 3sigma above background). The sensor sensitivity was not limited by the ability of the sensor to resolve the amperometric signal, but rather the capability of the fluidic dispenser to deliver analyte in a reproducible and quantitative fashion at volumes below 6.5 pL. A linear calibration curve of the charge transferred and the amount of injected lactate in the range between 65 and 266 fmol was obtained. For the study of lactate measurements from single heart cells, the biocompatability of various materials used in the electrochemical devices was evaluated. The biocompatibility of SU-8 was demonstrated. It was shown that direct contact of the strongly oxidizing AgCl layer with a single myocyte could cause the myocyte to promptly die. Therefore, a two-electrode configuration, with a platinized working electrode and a combined reference and counter platinum electrode, acting as a pseudo-reference electrode, was also used for single-cell measurements. On the basis of the theory of lactate metabolism and the anoxia model, the dynamic electrochemical measurements of lactate from healthy and anoxic single heart cells were obtained. The lactate content after metabolic inhibition was approximately three times that of the unpoisoned cell. The efflux of high level lactate was measured in real time after the injection of FCCP at high concentration at 150 ~ 200 muM. Whereas, the lactate content of cells poisoned by contact with the AglAgCl was similar to that of a healthy cell. The lactate signal produced from cells, inserted using drawn pipettes, was higher for cells with higher metabolic rate than cells that were quiescent and weakly contracting. In addition to standard photolithography techniques, the use of SU-8 and PDMS, as well as "soft lithography" techniques were explored for microsensor arrays and microfluidics applications. An integrated microfluidic biosensor chip and on-line flow injection analysis (FIA) monitoring system was designed. In summary, this thesis describes a generic method of fabricating a single cell sensor employing pL-scale volume and based on oxidase-reductase enzymes and microfluidics in a lab-on-a-chip format. The next technological advancement would be the immobilisation of enzymes on the microelectrodes, leading to improve stability and avoiding expensive enzyme strategies as a means to every response in the future. In the future, integration of individual enzyme sensor arrays and fluorescence sensors needs to be performed for the simultaneous detection of various biomolecules from a single cell

Individual Differences in the Speed of Facial Emotion Recognition Show Little Specificity but Are Strongly Related with General Mental Speed: Psychometric, Neural and Genetic Evidence

Facial identity and facial expression processing are crucial socio-emotional abilities but seem to show only limited psychometric uniqueness when the processing speed is considered in easy tasks. We applied a comprehensive measurement of processing speed and contrasted performance specificity in socio-emotional, social and non-social stimuli from an individual differences perspective. Performance in a multivariate task battery could be best modeled by a general speed factor and a first-order factor capturing some specific variance due to processing emotional facial expressions. We further tested equivalence of the relationships between speed factors and polymorphisms of dopamine and serotonin transporter genes. Results show that the speed factors are not only psychometrically equivalent but invariant in their relation with the Catechol-O Methyl-Transferase (COMT) Val158Met polymorphism. However, the 5-HTTLPR/rs25531 serotonin polymorphism was related with the first-order factor of emotion perception speed, suggesting a specific genetic correlate of processing emotions. We further investigated the relationship between several components of event-related brain potentials with psychometric abilities, and tested emotion specific individual differences at the neurophysiological level. Results revealed swifter emotion perception abilities to go along with larger amplitudes of the P100 and the Early Posterior Negativity (EPN), when emotion processing was modeled on its own. However, after partialling out the shared variance of emotion perception speed with general processing speed-related abilities, brain-behavior relationships did not remain specific for emotion. Together, the present results suggest that speed abilities are strongly interrelated but show some specificity for emotion processing speed at the psychometric level. At both genetic and neurophysiological levels, emotion specificity depended on whether general cognition is taken into account or not. These findings keenly suggest that general speed abilities should be taken into account when the study of emotion recognition abilities is targeted in its specificity

Label-free microfluidic paper-based electrochemical aptasensor for ultrasensitive and simultaneous multiplexed detection of cancer biomarkers

Simultaneous detection of multiple tumor biomarkers in body fluids could facilitate early diagnosis of lung cancer, so as to provide scientific reference for clinical treatment. This paper depicted a multi-parameter paper-based electrochemical aptasensor for simultaneous detection of carcinoembryonic antigen (CEA) and neuron-specific enolase (NSE) in a clinical sample with high sensitivity and specificity. The paper-based device was fabricated through wax printing and screen-printing, which enabled functions of sample filtration and sample auto injection. Amino functional graphene (NG)-Thionin (THI)- gold nanoparticles (AuNPs) and Prussian blue (PB)- poly (3,4- ethylenedioxythiophene) (PEDOT)- AuNPs nanocomposites were synthesized respectively. They were used to modify the working electrodes not only for promoting the electron transfer rate, but also for immobilization of the CEA and NSE aptamers. A label-free electrochemical method was adopted, enabling a rapid simple point-of-care testing. Experimental results showed that the proposed multi-parameter aptasensor exhibited good linearity in ranges of 0.01-500 ng mL for CEA (R  = 0.989) and 0.05-500 ng mL for NSE (R  = 0.944), respectively. The limit of detection (LOD) was 2 pg mL for CEA and 10 pg mL for NSE. In addition, the device was evaluated using clinical serum samples and received a good correlation with large electrochemical luminescence (ECL) equipment, which would offer a new platform for early cancer diagnostics, especially in those resource-limit areas

Genome-wide identification of the heat shock transcription factor gene family in two kiwifruit species

High temperatures have a significant impact on plant growth and metabolism. In recent years, the fruit industry has faced a serious threat due to high-temperature stress on fruit plants caused by global warming. In the present study, we explored the molecular regulatory mechanisms that contribute to high-temperature tolerance in kiwifruit. A total of 36 Hsf genes were identified in the A. chinensis (Ac) genome, while 41 Hsf genes were found in the A. eriantha (Ae) genome. Phylogenetic analysis revealed the clustering of kiwifruit Hsfs into three distinct groups (groups A, B, and C). Synteny analysis indicated that the expansion of the Hsf gene family in the Ac and Ae genomes was primarily driven by whole genome duplication (WGD). Analysis of the gene expression profiles revealed a close relationship between the expression levels of Hsf genes and various plant tissues and stress treatments throughout fruit ripening. Subcellular localization analysis demonstrated that GFP-AcHsfA2a/AcHsfA7b and AcHsfA2a/AcHsfA7b -GFP were localized in the nucleus, while GFP-AcHsfA2a was also observed in the cytoplasm of Arabidopsis protoplasts. The results of real-time quantitative polymerase chain reaction (RT-qPCR) and dual-luciferase reporter assay revealed that the majority of Hsf genes, especially AcHsfA2a, were expressed under high-temperature conditions. In conclusion, our findings establish a theoretical foundation for analyzing the potential role of Hsfs in high-temperature stress tolerance in kiwifruit. This study also offers valuable information to aid plant breeders in the development of heat-stress-resistant plant materials

In vivo microelectrode arrays for detecting multi-region epileptic activities in the hippocampus in the latent period of rat model of temporal lobe epilepsy

Temporal lobe epilepsy (TLE) is a form of refractory focal epilepsy, which includes a latent period and a chronic period. Microelectrode arrays capable of multi-region detection of neural activities are important for accurately identifying the epileptic focus and pathogenesis mechanism in the latent period of TLE. Here, we fabricated multi-shank MEAs to detect neural activities in the DG, hilus, CA3, and CA1 in the TLE rat model. In the latent period in TLE rats, seizures were induced and changes in neural activities were detected. The results showed that induced seizures spread from the hilus and CA3 to other areas. Furthermore, interneurons in the hilus and CA3 were more excited than principal cells and exhibited rhythmic oscillations at approximately 15 Hz in grand mal seizures. In addition, the power spectral density (PSD) of neural spikes and local field potentials (LFPs) were synchronized in the frequency domain of the alpha band (9–15 Hz) after the induction of seizures. The results suggest that fabricated MEAs have the advantages of simultaneous and precise detection of neural activities in multiple subregions of the hippocampus. Our MEAs promote the study of cellular mechanisms of TLE during the latent period, which provides an important basis for the diagnosis of the lesion focus of TLE

Exploring retinal ganglion cells encoding to multi-modal stimulation using 3D microelectrodes arrays

Microelectrode arrays (MEA) are extensively utilized in encoding studies of retinal ganglion cells (RGCs) due to their capacity for simultaneous recording of neural activity across multiple channels. However, conventional planar MEAs face limitations in studying RGCs due to poor coupling between electrodes and RGCs, resulting in low signal-to-noise ratio (SNR) and limited recording sensitivity. To overcome these challenges, we employed photolithography, electroplating, and other processes to fabricate a 3D MEA based on the planar MEA platform. The 3D MEA exhibited several improvements compared to planar MEA, including lower impedance (8.73 ± 1.66 kΩ) and phase delay (−15.11° ± 1.27°), as well as higher charge storage capacity (CSC = 10.16 ± 0.81 mC/cm2), cathodic charge storage capacity (CSCc = 7.10 ± 0.55 mC/cm2), and SNR (SNR = 8.91 ± 0.57). Leveraging the advanced 3D MEA, we investigated the encoding characteristics of RGCs under multi-modal stimulation. Optical, electrical, and chemical stimulation were applied as sensory inputs, and distinct response patterns and response times of RGCs were detected, as well as variations in rate encoding and temporal encoding. Specifically, electrical stimulation elicited more effective RGC firing, while optical stimulation enhanced RGC synchrony. These findings hold promise for advancing the field of neural encoding

A new approach to bias correction in RNA-Seq

Motivation: Quantification of sequence abundance in RNA-Seq experiments is often conflated by protocol-specific sequence bias. The exact sources of the bias are unknown, but may be influenced by polymerase chain reaction amplification, or differing primer affinities and mixtures, for example. The result is decreased accuracy in many applications, such as de novo gene annotation and transcript quantification

Paper-based microfluidic aptasensors

For in-situ disease markers detection, point-of-care (POC) diagnosis has great advantages in speed and cost compared with traditional techniques. The rapid diagnosis, prognosis, and surveillance of diseases can significantly reduce disease-related mortality and trauma. Therefore, increasing attention has been paid to the POC diagnosis devices due to their excellent diagnosis speed and portability. Over the past ten years, paper-based microfluidic aptasensors have emerged as a class of critical POC diagnosis devices and various aptasensors have been proposed to detect various disease markers. However, most aptasensors need further improvement before they can actually enter the market and be widely used. There is thus an urgent need to sort out the key points of preparing the aptasensors and the direction that needs to be invested in. This review summarizes the representative articles in the development of paper-based microfluidic aptasensors. These works can be divided into paper-based optical aptasensors and paper-based electrochemical aptasensors according to their output signals. Significant focus is applied to these works according to the following three parts: (1) The ingenious design of device structure; (2) Application and synthesis of nanomaterial; (3) The detection principle of the proposed aptasensor. This is a detailed and comprehensive review of paper-based microfluidic aptasensors. The accomplishments and shortcomings of the current aptasensors are outlined, the development direction and the future prospective are given. It is hoped that the research in this review can provide a reference for further development of more advanced, more effective paper-based microfluidic aptasensors for POC disease markers diagnosi

Low sample volume origami-paper-based graphene-modified aptasensors for label-free electrochemical detection of cancer biomarker-EGFR

In this work, an electrochemical paper-based aptasensor was fabricated for label-free and ultrasensitive detection of epidermal growth factor receptor (EGFR) by employing anti-EGFR aptamers as the bio-recognition element. The device used the concept of paper-folding, or origami, to serve as a valve between sample introduction and detection, so reducing sampling volumes and improving operation convenience. Amino-functionalized graphene (NH2-GO)/thionine (THI)/gold particle (AuNP) nanocomposites were used to modify the working electrode not only to generate the electrochemical signals, but also to provide an environment conducive to aptamer immobilization. Electrochemical characterization revealed that the formation of an insulating aptamer–antigen immunocomplex would hinder electron transfer from the sample medium to the working electrode, thus resulting in a lower signal. The experimental results showed that the proposed aptasensor exhibited a linear range from 0.05 to 200 ngmL−1 (R2 = 0.989) and a detection limit of 5 pgmL−1 for EGFR. The analytical reliability of the proposed paper-based aptasensor was further investigated by analyzing serum samples, showing good agreement with the gold-standard enzyme-linked immunosorbent assa