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.

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.

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.

Ultra-low level detection of hepatocellular carcinoma global methylation using a AuNP modified carbon fiber microelectrode.

Hepatocellular carcinoma (HCC) is one of the most common cancerous diseases, with a low 5 year survival rate. Global hypomethylation drives genomic instability, which is regarded as one biomarker for early diagnosis. Long interspersed nucleotide element-1 (LINE-1) makes up around 17% of the genome, and could be regarded as a surrogate marker for global DNA methylation. In this work, a gold nanoparticle (AuNP) modified carbon fiber microelectrode (CFME) with a diameter of 7 µm was applied for the first time to detect the methylation level of LINE-1, by distinguishing adsorption affinities between different DNA bases and AuNPs. Several parameters, including AuNP electrodeposition time, sample adsorption time, and DNA concentration have been analyzed and optimized. The detection limit of our assay was 0.1 nM with only 2 µL sample solution. And the CFME had an excellent sensitivity of 10% methylation change and had the capacity to distinguish only one methylated CpG site. The global DNA methylation level of real samples including cell lines and clinical tissues was tested. Higher signals of HCC cell lines and cancer tissues were observed respectively, compared with normal hepatic cell lines and normal tissues. This work provides a promising approach for HCC early diagnosis and prognosis.

A simple and low-cost screen printed electrode for hepatocellular carcinoma methylation detection.

There is a great demand for robust diagnostic and prognostic approaches for Hepatocellular Carcinoma (HCC). DNA methylation, a common epigenetic modification, has been found in many promoter regions of tumor suppressor genes. Hypermethylation of these gene promoters will repress the gene transcription and lead to the occurrence of cancers. The abnormal methyation level of the p16 gene promoter could be a promising marker for the detection of HCC. The adsorption affinities between different DNA bases and AuNPs are not the same. After bisulfite treatment and asymmetric PCR, methylation and unmethylation sequences can be changed into guanine-enriched and adenine-enriched sequences, respectively. A home-made gold nanoparticle modified screen printed carbon electrode (AuNP-SPCE) was employed to distinguish the adsorption affinities between guanine-enriched and adenine-enriched sequences, which could be used to analyze the level of DNA methylation. Several key experimental factors were investigated and optimized. The results had shown that the optimal AuNP electrodeposition time was 100 s and 15 min of adsorption could distinguish guanine-enriched and adenine-enriched sequences with a concentration of 100 nM at 25 °C. The detection limit of our AuNP-SPCE was 1.1 ng, and the assay had a good sensitivity of 10% methylation change and was able to distinguish only one methylated CpG site. What's more, the RSD over three assays with a disposable AuNP-SPCE was ≤7.2%. The assay was applied to real samples including cell lines and clinical tissues. Compared with normal hepatic cell lines and normal tissues, lower signals of HCC cell lines and cancer tissues were observed, respectively. It had shown a good discrimination of the abnormal methylation level of the p16 gene promoter.

Layer-by-layer chitosan-decorated pristine graphene on screen-printed electrodes by one-step electrodeposition for non-enzymatic hydrogen peroxide sensor.

Layer-by-layer chitosan-decorated pristine graphene on screen-printed electrodes was achieved by one-step electrodeposition method and an enzyme-free hydrogen peroxide electrochemical biosensor was fabricated for its application. Negatively charged pristine graphene absorbed with positively charged chitosan by electrostatic interactions was electrodeposited on the electrodes. The successful immobilization of pristine graphene was confirmed by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), Raman spectroscopy, X-ray photoelectron spectroscopy (XPS) and electrochemical characterizations. The graphene-chitosan volume ratio and cyclic voltammetry scan cycles during the electrodeposition process were optimized. The resulting hydrogen peroxide biosensors showed a 178 times improved sensitivity and exhibited two wide linear detection ranges from 20 µM to 20 mM and from 20 mM to 60 mM. The biosensors also showed a good reproducibility, stability, selectivity and could be used in real samples detection. The superior electrochemical performance of graphene-chitosan was attributed to the preservation of excellent properties of pristine graphene, and the formation of layer-by-layer structure with high surface area. The proposed strategy of direct immobilization of pristine graphene can be extended for the immobilization of other nanomaterials and biomolecules.

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.

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.

Optical fiber LPG biosensor integrated microfluidic chip for ultrasensitive glucose detection.

An optical fiber sensor integrated microfluidic chip is presented for ultrasensitive detection of glucose. A long-period grating (LPG) inscribed in a small-diameter single-mode fiber (SDSMF) is employed as an optical refractive-index (RI) sensor. With the layer-by-layer (LbL) self-assembly technique, poly (ethylenimine) (PEI) and poly (acrylic acid) (PAA) multilayer film is deposited on the SDSMF-LPG sensor for both supporting and signal enhancement, and then a glucose oxidase (GOD) layer is immobilized on the outer layer for glucose sensing. A microfluidic chip for glucose detection is fabricated after embedding the SDSMF-LPG biosensor into the microchannel of the chip. Experimental results reveal that the SDSMF-LPG biosensor based on such a hybrid sensing film can ultrasensitively detect glucose concentration as low as 1 nM. After integration into the microfluidic chip, the detection range of the sensor is extended from 2 µM to 10 µM, and the response time is remarkablely shortened from 6 minutes to 70 seconds.

Helminth protein vaccine induced follicular T helper cell for enhancement of humoral immunity against Schistosoma japonicum.

Protein vaccines combined with adjuvants have been widely used to induce immune responses, especially the humoral immune response, against molecular targets including parasites. Follicular T helper (Tfh) cells are the specialized providers of B-cell help, however, the induction of Tfh cells in protein vaccination has been rarely studied. Here, we report that the Schistosoma japonicum recombinant protein (SjGST-32) combined with tacrolimus (FK506) augmented the induction of Tfh cells, which expressed the canonical markers CXCR5, BCL6, and IL-21, and enhanced the humoral immune responses in BALB/c mice. Furthermore, the expression of IL-21R on germinal center (GC) B cells and memory B cells increased in immunized mice, which indicated that IL-21 from the induced Tfh cells interacted with IL-21R for activation of B cells and maintenance of long-lived humoral immunity. Our results suggest that helminth protein vaccine combined with FK506 induces Tfh cell for stimulating humoral immune responses and inducing long-lived humoral immunity.