Periodic magnetic modulation enhanced electrochemical analysis for highly sensitive determination of genomic DNA methylation.

DNA methylation aberrations have a strong correlation with cancer in early detection, diagnosis, and prognosis, which make them possible candidate biomarkers. Electrochemical biosensors offer rapid protocols for detecting DNA methylation status with minimal pretreatment of samples. However, the inevitable presence of background current in the time domain, including electrochemical noise and variations, limits the detection performance of these biosensors, especially for low concentration analytes. Here, we propose an ultrasensitive frequency-domain electrochemical analysis strategy to effectively separate the weak signals from background current. To achieve this, we employed periodic magnetic field modulation of magnetic beads (MBs) on and off the electrode surface to generate a periodic electrochemical signal for subsequent frequency-domain analysis. By capturing labeled MBs with as low as 0.5 pg of DNA, we successfully demonstrated a highly sensitive electrochemical method for determination of genome-wide DNA methylation levels. We also validated the effectiveness of this methodology using DNA samples extracted from three types of hepatocellular carcinoma (HCC) cell lines. The results revealed varying genomic methylation levels among different HCC cell lines, indicating the potential application of this approach for early-stage cancer detection in terms of DNA methylation status.

In situ continuously monitoring of cancer cell invasion process based on impedance sensing.

Activation of invasion and metastasis is recognized as one of the hallmarks of cancer. There are 90% of cancer-related deaths due to metastasis and given that it is worthy of note to study cancer progression and metastasis. Owing to restricted tools used to underpin the study of tumor invasion process, an on-site platform was developed to monitor this event in vitro. We used interdigital gold electrodes to monitor the dynamic process of cancer cells invading into extracellular matrix in situ continuously. Influences of collagen concentration and number of cancer cells on the measured impedance was exhibited. In addition, the parameters used to demonstrate the experiment results were optimized. The change of impedance magnitude indicated the cell-matrix interaction during invasion process. The potential further use of this platform would be complementary in cell studies when concerning metastasis.

The label-free separation and culture of tumor cells in a microfluidic biochip.

Circulating tumor cells (CTCs) from liquid biopsy have shown a strong correlation to the clinical outcome of cancer patients. The enumeration and cytological analysis of CTCs have attracted increasing efforts for cancer disease management amid immunotherapy and personalized medicine. However, both enumeration and cytological analysis are challenging due to the rarity of CTCs and the lack of integrated solutions for the minimal risk of cell loss in the course of CTC procurement. We report a simple microfluidic chip permitting a one-stop solution for streamlining the on-chip cell separation, capture, immunofluorescence assay and/or in situ culture of isolated cells devoid of risky manual steps. Our results showed effective trapping of single cells, doublets and cell lumps isolated from blood in the same device. On-chip immunostaining revealed normal cell morphology and the characterization of cell expansion uncovered an altered cell growth curve with a reduced lag phase as compared to the conventional culture despite closely matching cell growth rates. The cells were viable and functional for as long as 11 days inside our chip and cell migration was also readily observed, with lumps showing greater aggressiveness than single cells. With these results, we expect promising applications of our one-stop solution for liquid biopsy via CTCs.

Semantic and Generalized Entropy Loss Functions for Semi-Supervised Deep Learning.

The increasing size of modern datasets combined with the difficulty of obtaining real label information (e.g., class) has made semi-supervised learning a problem of considerable practical importance in modern data analysis. Semi-supervised learning is supervised learning with additional information on the distribution of the examples or, simultaneously, an extension of unsupervised learning guided by some constraints. In this article we present a methodology that bridges between artificial neural network output vectors and logical constraints. In order to do this, we present a semantic loss function and a generalized entropy loss function (Rényi entropy) that capture how close the neural network is to satisfying the constraints on its output. Our methods are intended to be generally applicable and compatible with any feedforward neural network. Therefore, the semantic loss and generalized entropy loss are simply a regularization term that can be directly plugged into an existing loss function. We evaluate our methodology over an artificially simulated dataset and two commonly used benchmark datasets which are MNIST and Fashion-MNIST to assess the relation between the analyzed loss functions and the influence of the various input and tuning parameters on the classification accuracy. The experimental evaluation shows that both losses effectively guide the learner to achieve (near-) state-of-the-art results on semi-supervised multiclass classification.

A flexible cell concentrator using inertial focusing.

Cell concentration adjustment is intensively implemented routinely both in research and clinical laboratories. Centrifuge is the most prevalent technique for tuning biosample concentration. But it suffers from a number of drawbacks, such as requirement of experienced operator, high cost, low resolution, variable reproducibility and induced damage to sample. Herein we report on a cost-efficient alternative using inertial microfluidics. While the majority of existing literatures concentrate on inertial focusing itself, we identify the substantial role of the outlet system played in the device performance that has long been underestimated. The resistances of the outlets virtually involve in defining the cutoff size of a given inertial filtration channel. Following the comprehensive exploration of the influence of outlet system, we designed an inertial device with selectable outlets. Using both commercial microparticles and cultured Hep G2 cells, we have successfully demonstrated the automated concentration modification and observed several key advantages of our device as compared with conventional centrifuge, such as significantly reduced cell loss (only 4.2% vs. ~40% of centrifuge), better preservation of cell viability and less processing time as well as the increased reproducibility due to absence of manual operation. Furthermore, our device shows high effectiveness for concentrated sample (e.g., 1.8 × 106 cells/ml) as well. We envision its promising applications in the circumstance where repetitive sample preparation is intensely employed.

Study of the union method of microelectrode array and AFM for the recording of electromechanical activities in living cardiomyocytes.

Electrophysiology and mechanics are two essential components in the functions of cardiomyocytes and skeletal muscle cells. The simultaneous recording of electrophysiological and mechanical activities is important for the understanding of mechanisms underlying cell functions. For example, on the one hand, mechanisms under cardiovascular drug effects will be investigated in a comprehensive way by the simultaneous recording of electrophysiological and mechanical activities. On the other hand, computational models of electromechanics provide a powerful tool for the research of cardiomyocytes. The electrical and mechanical activities are important in cardiomyocyte models. The simultaneous recording of electrophysiological and mechanical activities can provide much experimental data for the models. Therefore, an efficient method for the simultaneous recording of the electrical and mechanical data from cardiomyocytes is required for the improvement of cardiac modeling. However, as far as we know, most of the previous methods were not easy to be implemented in the electromechanical recording. For this reason, in this study, a union method of microelectrode array and atomic force microscope was proposed. With this method, the extracellular field potential and beating force of cardiomyocytes were recorded simultaneously with a low root-mean-square noise level of 11.67 µV and 60 pN. Drug tests were conducted to verify the feasibility of the experimental platform. The experimental results suggested the method would be useful for the cardiovascular drug screening and refinement of the computational cardiomyocyte models. It may be valuable for exploring the functional mechanisms of cardiomyocytes and skeletal muscle cells under physiological or pathological conditions.

Expanded Carbon Nanotube Fiber at the Liquid-Air Interface for High-Performance Fiber-Based Supercapacitors and Electrochemical Sensors.

Carbon nanotube fibers (CNTFs) are widely utilized in flexible and wearable electronics due to their outstanding electrical and mechanical properties. However, the spinning process of CNTFs has limited the CNTs from exposure, leading to an ultralow usage efficiency of individual CNTs. Here, we propose an electrochemical expansion strategy of a single CNTF at the liquid-air interface, forming a macroscopic spindle-shaped CNTF (SS-CNTF) with an enlarged volume of up to 5000-fold upon the spindle. The obtained spindle-shaped structure endows CNTF with a high specific surface area together with excellent conductivity and good mechanical properties. Therefore, the SS-CNTF-based devices exhibit outstanding performances both in energy storage (electrical double-layer supercapacitor, energy density: 11.22 Wh kg-1, power density: 203.9 kW kg-1) and electrochemical sensing (ascorbic acid: 1.26 µA µM-1 cm-2; dopamine: 103.91 µA µM-1 cm-2; uric acid: 11.53 µA µM-1 cm-2). The novel architecture of SS-CNTF prepared by one-step electrochemical expansion at the liquid-air interface enabled its high performance in multiple applications, providing new insight into the development of CNTF-based devices.

Multi-Parameter Detection of Urine Based on Electropolymerized PANI: PSS/AuNPs/SPCE.

Urine analysis is widely used in clinical practice to indicate human heathy status and is important for diagnosing chronic kidney disease (CKD). Ammonium ions (NH4+), urea, and creatinine metabolites are main clinical indicators in urine analysis of CKD patients. In this paper, NH4+ selective electrodes were prepared using electropolymerized polyaniline-polystyrene sulfonate (PANI: PSS), and urea- and creatinine-sensing electrodes were prepared by modifying urease and creatinine deiminase, respectively. First, PANI: PSS was modified on the surface of an AuNPs-modified screen-printed electrode, as a NH4+-sensitive film. The experimental results showed that the detection range of the NH4+ selective electrode was 0.5~40 mM, and the sensitivity reached 192.6 mA M-1 cm-2 with good selectivity, consistency, and stability. Based on the NH4+-sensitive film, urease and creatinine deaminase were modified by enzyme immobilization technology to achieve urea and creatinine detection, respectively. Finally, we further integrated NH4+, urea, and creatinine electrodes into a paper-based device and tested real human urine samples. In summary, this multi-parameter urine testing device offers the potential for point-of-care testing of urine and benefits the efficient chronic kidney disease management.

Magnetic Immunosensor Coupled to Enzymatic Signal for Determination of Genomic DNA Methylation.

Aberrations of genomic DNA methylation have been confirmed to be involved in the evolution of human cancer and have thus gained the potential to be depicted as biomarkers for cancer diagnostics and prognostic predictions, which implicates an urgent need for detection of total genomic DNA methylation. In this work, we suggested an assay for the quantification of global DNA methylation, utilizing methylation specific antibody (5mC) modified magnetic beads (MBs) for immunorecognition and affinity enrichment. Subsequently, the captured DNA on the surface of MBs interacted with the glucose oxidase-conjugated DNA antibody whose catalytic reaction product was engaged in electrochemical detection of the overall level of DNA methylation on a PB-doped screen-printed electrode. With 15 pg of input DNA, which, to our best knowledge, is the lowest required amount of DNA without sodium bisulfite treatment or amplification, this test strategy was able to perceive as low as 5% methylation level within 70 min including the preparation of anti-5mC-MBs. We believe this detection technique offers a promising option to detect global DNA methylation in both academic and clinical scenarios.

Tissue-like Conductive Ti3C2/Sodium Alginate Hybrid Hydrogel for Electrochemical Sensing.

Endowed with a soft and conductive feature, hydrogels have been widely used as interface materials in bioelectronics to fulfill mechanical matching and bidirectional exchange between electronic platforms and living samples. Despite their ionic conductivity, the lack of electron mobility has limited their further applications in biosensing, especially in the field of electrochemical sensing. Here, we propose a Ti3C2/sodium alginate (SA) hybrid hydrogel with not only a tissue-like mechanical strength (down to 80 kPa) but also a combined exchange interface for ions and electrons, realizing both mechanical and electrical coupling toward biological tissues. Due to the shared gelation tendency with cations, the Ti3C2 sheets and SA chains can be easily in situ coassembled through a one-step electrogelation method, making the hybrid hydrogel a well-suited interface layer for device functionalization. In addition, the typical two-dimensional (2D) structure and the abundant active terminals of Ti3C2 have endowed the Ti3C2/SA with a massive loading capacity toward catalytic nanoparticles. For example, the Prussian Blue (PB)-loaded Ti3C2/SA hybrid hydrogel exhibited an excellent electrochemical performance (sensitivity: 600 nA µM-1 cm-2; LOD: 12 nM) toward hydrogen peroxide sensing in tissue fluids, illustrating a promising application potentiality of the hybrid hydrogel in biochemical detection at tissue interfaces.

A PCR-free screen-printed magnetic electrode for the detection of circular RNA from hepatocellular cancer based on a back-splice junction.

Circular RNA (circRNA) has the potential to be applied to disease diagnosis and therapy. However, the currently available circRNA detection techniques are limited. This work proposes a sensitive and selective approach for circRNA detection based on gold nanoparticle-modified screen-printed magnetic electrodes (AuNPs-SPME). Magnetic beads (MBs) with capture probes based on specific back-splice junction (BSJ) sites were employed to identify and selectively isolate the target circRNA, which could be directly adsorbed onto the AuNPs-SPME. Then, the circRNA attached to the surface was detected by changes in the methylene blue redox signal. The simple and time-saving AuNPs-SPME is highly sensitive (LOD = 1.0 pM) to circCDYL, one of the biomarkers of hepatocellular cancer (HCC). The analytical performance of the method presented has also been verified in human serum samples, holding great promise for clinical diagnosis.

An integrated electrochemical POCT platform for ultrasensitive circRNA detection towards hepatocellular carcinoma diagnosis.

Hepatocellular carcinoma (HCC) is the fourth leading cause of cancer-related death. Circ-CDYL, one of the circular RNAs (circRNAs), is recognized as an independent marker for HCC early diagnosis. Point-of-care testing (POCT) of circRNA is essential and in great demand for clinical applications. Herein, we report a fully integrated electrochemical POCT platform for circRNA detection based on Au nanoflowers (AuNFs)/peptide nucleic acid (PNA) modified carbon-fiber microelectrode (CFME). PNA is applied as the recognition element, highly specified for a back-splice junction of circRNA. AuNFs increased active site for PNA probes, improving target-capturing efficiency at an ultralow level. The platform provides a linear range of 10 fM to 1 µM, with a detection limit as low as 3.29 fM. This biosensor demonstrates high specificity towards one-base mismatch and is stable for up to 24 days. The analytical performance has also been verified in human serum samples, demonstrating the potential utility in clinical POCT applications for HCC.

3D bimetallic Au/Pt nanoflowers decorated needle-type microelectrode for direct in situ monitoring of ATP secreted from living cells.

Adenosine triphosphate (ATP) plays a crucial role in energy metabolism and extracellular purinergic signaling. A 3D bimetallic Au/Pt nanoflowers decorated ATP microelectrode biosensor prepared by facile and effective template-free electrodeposition was firstly reported, realizing local detection of cellular ATP secretion. The ATP biosensor was developed by co-immobilization of glucose oxidase and hexokinase, exhibiting long-term stability (79.39 ± 9.15% of its initial value remained after 14 days at 4 °C) and high selectivity with a limit of detection down to 2.5 µM (S/N = 3). The resulting ATP biosensor was then used for direct in situ monitoring of ATP secreted from living cells (PC12) with the stimulation of high K+ solutions. The obtained current was about 21.6 ± 3.4 nA (N = 6), corresponding to 12.2 ± 2.8 µM ATP released from cells, right in the micromolar range and consistent with the suggested levels. The 3D bimetallic Au/Pt nanoflowers possess excellent catalytic activity and large electroactive surface area, contributing to enzymatic activity preservation and long-term stability. This work provides a promising platform for long-time monitoring of other neurotransmitters and secretions in cellular glycolysis and apoptosis processes in the future.

Isolation of cells from whole blood using shear-induced diffusion.

Extraction of cells of interest directly from whole blood is in high demand, yet extraordinary challenging due to the complex hemodynamics and hemorheology of the sample. Herein, we describe a new microfluidic platform that exploits the intrinsic complex properties of blood for continuous size-selective focusing and separation of cells directly from unprocessed whole blood. The novel system only requires routinely accessible saline solution to form a sandwiched fluid configuration and to initiate a strong effect of shear-induced diffusion of cells, which is coupled with fluid inertia for effective separation. Separations of beads and cells from whole blood have been successfully demonstrated with high efficiency (89.8%) at throughput of 6.75 mL/hr (106-107 cells/s) of whole blood. Rapid isolation of circulating tumor cells (CTCs) from peripheral blood sample of hepatocarcinoma patients is also shown as a proof of principle.

Efficient detection of internal infestation in wheat based on biophotonics.

In the process of grain storage, there are many losses of grain quantity and quality for the sake of insects. As a result, it is necessary to find a rapid and economical method for detecting insects in the grain. The paper innovatively proposes a model of detecting internal infestation in wheat by combining pattern recognition and BioPhoton Analytical Technology (BPAT). In this model, the spontaneous ultraweak photons emitted from normal and insect-contaminated wheat are firstly measured respectively. Then, position, distribution and morphological characteristics can be extracted from the measuring data to construct wheat feature vector. Backpropagation (BP) neural network based on genetic algorithm is employed to take decision on whether wheat kernel has contaminated by insects. The experimental results show that the proposed model can differentiate the normal wheat from the insect-contaminated one at an average accuracy of 95%. The model can also offer a novel thought for detecting internal infestation in the wheat.

Study of laser uncaging induced morphological alteration of rat cortical neurites using atomic force microscopy.

Activity-dependent structural remodeling is an important aspect of neuronal plasticity. In the previous researches, neuronal structure variations resulting from external interventions were detected by the imaging instruments such as the fluorescence microscopy, the scanning/transmission electron microscopy (SEM/TEM) and the laser confocal microscopy. In this article, a new platform which combined the photochemical stimulation with atomic force microscopy (AFM) was set up to detect the activity-dependent structural remodeling. In the experiments, the cortical neurites on the glass coverslips were stimulated by locally uncaged glutamate under the ultraviolet (UV) laser pulses, and a calcium-related structural collapse of neurites (about 250 nm height decrease) was observed by an AFM. This was the first attempt to combine the laser uncaging with AFM in living cell researches. With the advantages of highly localized stimulation (<5 µm), super resolution imaging (<3.8 nm), and convenient platform building, this system was suitable for the quantitative observation of the neuron mechanical property variations and morphological alterations modified by neural activities under different photochemical stimulations, which would be helpful for studying physiological and pathological mechanisms of structural and functional changes induced by the biomolecule acting.