Combining sensors and actuators with electrowetting-on-dielectric (EWOD): advanced digital microfluidic systems for biomedical applications.

The concept of digital microfluidics (DMF) enables highly flexible and precise droplet manipulation at a picoliter scale, making DMF a promising approach to realize integrated, miniaturized "lab-on-a-chip" (LOC) systems for research and clinical purposes. Owing to its simplicity and effectiveness, electrowetting-on-dielectric (EWOD) is one of the most commonly studied and applied effects to implement DMF. However, complex biomedical assays usually require more sophisticated sample handling and detection capabilities than basic EWOD manipulation. Alternatively, combined systems integrating EWOD actuators and other fluidic handling techniques are essential for bringing DMF into practical use. In this paper, we briefly review the main approaches for the integration/combination of EWOD with other microfluidic manipulation methods or additional external fields for specified biomedical applications. The form of integration ranges from independently operating sub-systems to fully coupled hybrid actuators. The corresponding biomedical applications of these works are also summarized to illustrate the significance of these innovative combination attempts.

A Cell State Monitoring System with Integrated In Situ Imaging and pH Detection.

Cell models are one of the most widely used basic models in biological research, and a variety of in vitro cell culture techniques and models have been developed recently to simulate the physiological microenvironment in vivo. However, regardless of the technique or model, cell culture is the most fundamental but crucial component. As a result, we have developed a cell culture monitoring system to assess the functional status of cells within a biochip. This article focuses on a mini-microscope made from a readily available camera for in situ continuous observation of cell growth within a biochip and a pH sensor based on optoelectronic sensing for measuring pH. With the aid of this monitoring system, scientists can keep an eye on cell growth in real time and learn how the pH of the culture medium affects it. This study offers a new approach for tracking cells on biochips and serves as a valuable resource for enhancing cell culture conditions.

Microfluidic integrated capacitive biosensor for C-reactive protein label-free and real-time detection.

A microfluidic chip has been integrated with a capacitive biosensor based on mass-producible three-dimensional (3D) interdigital electrode arrays. To achieve the monitoring of biosensor preparation and cardiac- and periodontitis-related biomarkers, all the processes were detected in a continuously on-site way. Fabrication steps for the microfluidic chip-bonded 3D interdigital capacitor biosensor include gold thiol modification, the activation of EDC/sulfo-NHS, and the bioconjugation of antibodies. Fluorescent characterization and X-ray photoelectron spectroscopy analysis were applied to assess the successful immobilization of the C-reactive protein (CRP) antibody. The experimental results indicate the good specificity and high sensitivity of the microfluidic integrated 3D capacitive biosensor. The limit of detection of the 3D capacitive biosensor for CRP label-free detection was about 1 pg mL-1. This 3D capacitive biosensor with integrated microfluidics is mass-producible and has achieved the on-site continuous detection of cardiac- and periodontitis-related biomarkers with high performance.

A Novel Mass-Producible Capacitive Sensor with Fully Symmetric 3D Structure and Microfluidics for Cells Detection.

Affinity biosensors of interdigitated electrodes have been widely used in cell detection. This research presents a mass-producible and disposable three-dimensional (3D) structure capacitive sensor based on the integrated circuit package lead frames for cell concentration detection. The fully symmetric 3D interdigital electrode structure makes the sensor more homogeneous and sensitive. (3-Aminopropyl) triethoxysilane (APTES) and glutaraldehyde are immobilized onto gold-plated electrodes. By overlaying the microfluidic channels on top, the volume of the solution is kept constant to obtain repeatable measured capacitance values. Moreover, using the upgraded reading and writing functions and circular measurement of the E4980A Data Transfer Program, an automatic multigroup test system is developed. It is shown that the cell concentration and capacitance are inversely correlated, and the cell concentration range of 10³â»106 CFU∙mL-1 is achieved. In addition, the rate of capacitance change matches that of state-of-the-art biosensors reported. A program is developed to find the optimal voltage and frequency for linear fitting between the capacitance change and cell concentration. Future work will employ machine learning-based data analysis to drug resistance sensitivity test of cell lines and cell survival status.

Investigation of Controllable Nanoscale Heat-Denatured Bovine Serum Albumin Films on Graphene.

Two-dimensional graphene devices are widely used for biomolecule detection. Nevertheless, the surface modification of graphene is critical to achieve the high sensitivity and specificity required for biological detection. Herein, native bovine serum albumin (BSA) in inorganic solution is denatured on the graphene surface by heating, leading to the formation of nanoscale BSA protein films adsorbed on the graphene substrate via π-stacking interactions. This technique yields a controllable, scalable, uniform, and high-coverage method for graphene biosensors. Further, the application of such nanoscale heat-denatured BSA films on graphene as a universal graphene biosensor platform is explored. The thickness of heat-denatured BSA films increased with heating time and BSA concentration but decreased with solvent concentration as confirmed by atomic force microscopy. The noncovalent interaction between denatured BSA films and graphene was investigated by Raman spectroscopy. BSA can act as a p-type and n-type dopant by modulating pH-dependent net charges on the layered BSA-graphene surface, as assessed by current-voltage measurements. Chemical groups of denatured BSA films, including amino and carboxyl groups, were verified by X-ray photoelectron microscopy, attenuated total reflectance-Fourier transform infrared spectra, and fluorescent labeling. The tailoring of the BSA-graphene surfaces through chemical modification, controlled thickness, and doping type via noncovalent interactions provides a controllable, multifunctional biosensor platform for molecular diagnosis without the possibility of nonspecific adsorption on graphene.

Ultrasensitive Detection of Dual Cancer Biomarkers with Integrated CMOS-Compatible Nanowire Arrays.

A direct, rapid, highly sensitive and specific biosensor for detection of cancer biomarkers is desirable in early diagnosis and prognosis of cancer. However, the existing methods of detecting cancer biomarkers suffer from poor sensitivity as well as the requirement of enzymatic labeling or nanoparticle conjugations. Here, we proposed a two-channel PDMS microfluidic integrated CMOS-compatible silicon nanowire (SiNW) field-effect transistor arrays with potentially single use for label-free and ultrasensitive electrical detection of cancer biomarkers. The integrated nanowire arrays showed not only ultrahigh sensitivity of cytokeratin 19 fragment (CYFRA21-1) and prostate specific antigen (PSA) with detection to at least 1 fg/mL in buffer solution but also highly selectivity of discrimination from other similar cancer biomarkers. In addition, this method was used to detect both CYFRA21-1 and PSA real samples as low as 10 fg/mL in undiluted human serums. With its excellent properties and miniaturization, the integrated SiNW-FET device opens up great opportunities for a point-of-care test (POCT) for quick screening and early diagnosis of cancer and other complex diseases.

Facile Preparation of Bifunctional Monodisperse Nanospheres with Tunable Size and Luminescence.

Nanotechnology has found wide use in biomedical applications and the food and bioprocessing industry. In this light, we demonstrate a facile strategy to prepare bifunctional monodisperse silica nanospheres encapsulating chitosan-coated magnetic nanoparticles and CdTe quantum dots. The size of these composite spheres can be adjusted from 90 nm to 500 nm by varying the concentration of ammonia, water, tetraethyl orthosilicate, and the ratio of the chitosan-coated magnetic nanoparticles and CdTe quantum dots. The composite spheres are characterized using scanning electron microscope analyses, transmission electron microscope analyses, energy-dispersed spectrum studies, Malvern Zetasizer, vibrating sample magnetometer, and fluorescence microscopy. The spheres exhibit good monodispersion and favorable superparamagnetic and fluorescent properties. The luminescence of the spheres can be varied by using different types of coated quantum dots. Such composite spheres with tunable characteristics allow for external manipulation of research systems by magnetic fields together with the real-time fluorescent monitoring of multiple samples. The abovementioned properties can potentially be exploited for application in the biomedical and biosensing fields.

Pretreatment with wortmannin alleviates lipopolysaccharide/d-galactosamine-induced acute liver injury.

Intestinal endotoxemia-induced liver injury is a common clinical disease which leads to liver failure and death. Wortmannin, an inhibitor of phosphatidylinositol 3-kinase, could be used for suppressing autophagy in vitro and in vivo. Autophagy is an evolutionarily conserved and lysosome dependent protein degradation pathway, which participates in various physiological and pathological processes. The present study aims to explore the effect of pretreatment with wortmannin on acute liver injury and the autophagy in acute liver injury. We demonstrated that wortmannin could downregulate the expression of phosphorylated extracellular regulated protein kinase and p65, decrease the production and release of hepatic inflammatory cytokines, and then reduce hepatocytes apoptosis and necrosis. More importantly, we found that autophagy was induced to increase in LPS/D-GalN-induced acute liver injury, and pretreatment with wortmannin could effectively inhibit increased autophagy in acute liver injury. In conclusion, these results indicate that wortmannin plays a protective role in LPS/D-GalN induced hepatocytotoxity maybe by inhibiting autophagy and could be acted as a target for the treatment of acute liver injury.

Quantitative assessment of the influence of common variations (rs8034191 and rs1051730) at 15q25 and lung cancer risk.

Several genome-wide association studies on lung cancer (LC) have reported similar findings of a new susceptibility locus, 15q25. After that, a number of studies reported that rs8034191 and rs1051730 polymorphisms at chromosome 15q25 have been implicated in LC risk. However, studies have yielded contradictory results. To derive a more precise estimation of the relationship, a meta-analysis of 43,742 LC cases and 58,967 controls from 17 published case-control studies was performed. Overall, significantly elevated LC risk was associated with rs8034191-C (OR = 1.26, 95% CI 1.22-1.31, P < 10(-5)) and rs105173-A variant (OR = 1.28, 95% CI 1.20-1.36, P < 10(-5)) when all studies were pooled into the meta-analysis. In the subgroup analysis by ethnicity, significantly increased risks were found for rs8034191 and rs105173 polymorphisms among Caucasians and African American, while no significant associations were observed for the two polymorphisms in East Asians. In addition, we found that rs8034191 and rs105173 confer risk, for both adenocarcinoma and squamous cell carcinoma when stratified by histological types of LC. Furthermore, our results on stratified analysis according to smoking status showed an increased LC risk in ever-smokers, while no associations were detected among never-smokers for the two polymorphisms. In conclusion, this meta-analysis demonstrated that the two common variations (rs8034191 and rs1051730) at 15q25 are a risk factor associated with increased LC susceptibility, but these associations vary in different ethnic populations.

Rolling circle amplification immunoassay combined with gold nanoparticle aggregates for colorimetric detection of protein.

A highly sensitive and novel colorimetric rolling circle amplification (RCA) immunoassay for detecting C-reactive protein (CRP) has been developed. In the assay, a CRP capture antibody was immobilized on magnetic beads and a CRP detection antibody was conjugated with single-stranded DNA (ssDNA) using N-[ε-maleimidocaproyloxy] sulfosuccinimide ester. Along with the addition of CRP, a "sandwich" structure was formed. Subsequently, the ssDNA was used as a primer to initiate the RCA reaction in the presence of the circular template, phi29 DNA polymerase and deoxynucleotide triphosphates. The RCA product obtained by magnetic separation, and long tandem repeated sequences mediated the aggregation of gold nanoparticles (AuNPs), which could be observed by the naked eye or quantified using absorption spectra with a detection limit of 30 fg mL(-1) and a linear response range from 10 ng mL(-1) to 1 pg mL(-1). This assay offers the advantages of isothermal conditions, low cost and label-free quantification that could be hopeful for ultrasensitive and robust visual protein detection.

Signal-to-noise ratio enhancement of silicon nanowires biosensor with rolling circle amplification.

Herein, we describe a novel approach for rapid, label-free and specific DNA detection by applying rolling circle amplification (RCA) based on silicon nanowire field-effect transistor (SiNW-FET) for the first time. Highly responsive SiNWs were fabricated with a complementary metal oxide semiconductor (CMOS) compatible anisotropic self-stop etching technique which eliminated the need for hybrid method. The probe DNA was immobilized on the surface of SiNW, followed by sandwich hybridization with the perfectly matched target DNA and RCA primer that acted as a primer to hybridize the RCA template. The RCA reaction created a long single-stranded DNA (ssDNA) product and thus enhanced the electronic responses of SiNW significantly. The signal-to-noise ratio (SNR) as a figure-of-merit was analyzed to estimate the signal enhancement and possible detection limit. The nanosensor showed highly sensitive concentration-dependent conductance change in response to specific target DNA sequences. Because of the binding of an abundance of repeated sequences of RCA products, the SNR of >20 for 1 fM DNA detection was achieved, implying a detection floor of 50 aM. This RCA-based SiNW biosensor also discriminated perfectly matched target DNA from one-base mismatched DNA with high selectivity due to the substantially reduced nonspecific binding onto the SiNW surface through RCA. The combination of SiNW FET sensor with RCA will increase diagnostic capacity and the ability of laboratories to detect unexpected viruses, making it a potential tool for early diagnosis of gene-related diseases.

Non-synonymous single nucleotide polymorphisms in the P2X receptor genes: association with diseases, impact on receptor functions and potential use as diagnosis biomarkers.

P2X receptors are Ca2+-permeable cationic channels in the cell membranes, where they play an important role in mediating a diversity of physiological and pathophysiological functions of extracellular ATP. Mammalian cells express seven P2X receptor genes. Single nucleotide polymorphisms (SNPs) are widespread in the P2RX genes encoding the human P2X receptors, particularly the human P2X7 receptor. This article will provide an overview of the non-synonymous SNPs (NS-SNPs) that have been associated with or implicated in altering the susceptibility to pathologies or disease conditions, and discuss the consequences of the mutations resulting from such NS-SNPs on the receptor functions. Disease-associated NS-SNPs in the P2RX genes have been valuable in understanding the disease etiology and the receptor function, and are promising as biomarkers to be used for the diagnosis and development of stratified therapeutics.

Capacitive Bionic Magnetic Sensors Based on One-Step Biointerface Preparation.

Magnetic biomolecule-based bionic magnetic field sensors are anticipated to open up novel pathways for magnetic field detection. The detection range and accuracy of current bionic magnetic field sensors are limited, and little work is based on the capacitive response principle. We successfully developed a biochemical interface with an extralarge target-receptor size ratio, which can be manufactured in a single step for weak magnetic field detection across a wide frequency range, and we used electrochemical capacitance as a magnetic field change conduction strategy. The thickness-controllable nanoscale bovine serum albumin/graphene layer on an indium tin oxide working electrode combines with the one-step preparation method to immobilize the MagR/Cry4 complex. This capacitive bionic magnetic sensor can achieve the detection range of 0-120 mT. This biointerface design strategy obtains the further improvement of the performance of this bionic magnetic field sensor. Furthermore, the biointerface construction and optimization methodology in this proposal has potential applications in the design of other medical biosensors.

Monitoring of Sweat Ions and Physiological Parameters via a Reconfigurable Modular System.

In recent years, wearable sensors have revolutionized health monitoring by enabling continuous, real-time tracking of human health and performance. These noninvasive devices are usually designed to monitor human physical state and biochemical markers. However, enhancing their functionalities often demands intricate customization by designers and additional expenses for users. Here, we present a strategy using assembled modular circuits to customize health monitoring wearables. The modular circuits can be effortlessly reconfigured to meet various specific requirements, facilitating the incorporation of diverse functions at a lower cost. To validate this approach, modular circuits were employed to develop four distinct systems for in vitro evaluations. These systems enabled the detection of sweat biomarkers and physical signals under various scenarios, including sedentary state, exercise, and daily activities with or without incorporating iontophoresis to induce sweat. Four key sweat markers (K+, Ca2+, Na+, and pH) and three essential physical indicators (heart rate, blood oxygen levels, and skin temperature) are selected as the detection targets. Commercial methods were also used to evaluate the potential for effective health monitoring with our technique. This reconfigurable modular wearable (ReModuWear) system promises to provide more easy-to-use and comprehensive health assessments. Additionally, it may contribute to environmental sustainability by reusing modules.

Detecting EGFR gene amplification using a fluorescence in situ hybridization platform based on digital microfluidics.

Signal transduction mediated by epidermal growth factor receptor (EGFR) gene affects the proliferation, invasion, metastasis, and angiogenesis of tumor cells. In particular, non-small cell lung cancer (NSCLC) patients with increased in copy number of EGFR gene are often sensitive to tyrosine kinase inhibitors. Despite being the standard for detecting EGFR amplification in the clinic, fluorescence in situ hybridization (FISH) traditionally involves repetitive and complex benchtop procedures that are not only time consuming but also require well-trained personnel. To address these limitations, we develop a digital microfluidics-based FISH platform (DMF-FISH) that automatically implements FISH operations. This system mainly consists of a DMF chip for reagent operation, a heating array for temperature control and a signal processing system. With the capability of automatic droplet handling and efficient temperature control, DMF-FISH performs cell digestion, gradient elution, hybridization and DAPI staining without manual intervention. In addition to operational feasibility, DMF-FISH yields comparable performance with the benchtop FISH protocol but reducing the consumption of DNA probe by 87 % when tested with cell lines and clinical samples. These results highlight unique advantages of the fully automated DMF-FISH system and thus suggest its great potential for clinical diagnosis and personalized therapy of NSCLC.

Rapid automated extracellular vesicle isolation and miRNA preparation on a cost-effective digital microfluidic platform.

As a prerequisite for extracellular vesicle (EV) -based studies and diagnosis, effective isolation, enrichment and retrieval of EV biomarkers are crucial to subsequent analyses, such as miRNA-based liquid biopsy for non-small-cell lung cancer (NSCLC). However, most conventional approaches for EV isolation suffer from lengthy procedure, high cost, and intense labor. Herein, we introduce the digital microfluidic (DMF) technology to EV pretreatment protocols and demonstrate a rapid and fully automated sample preparation platform for clinical tumor liquid biopsy. Combining a reusable DMF chip technique with a low-cost EV isolation and miRNA preparation protocol, the platform completes automated sample processing in 20-30 min, supporting immediate RT-qPCR analyses on EV-derived miRNAs (EV-miRNAs). The utility and reliability of the platform was validated via clinical sample processing for EV-miRNA detection. With 23 tumor and 20 non-tumor clinical plasma samples, we concluded that EV-miR-486-5p and miR-21-5p are effective biomarkers for NSCLC with a small sample volumn (20-40 µL). The result was consistent to that of a commercial exosome miRNA extraction kit. These results demonstrate the effectiveness of DMF in EV pretreatment for miRNA detection, providing a facile solution to EV isolation for liquid biopsy.

A colorimetric method for H1N1 DNA detection using rolling circle amplification.

A highly sensitive and specific colorimetry-based rolling circle amplification (RCA) assay has been successfully developed as a method for the effective detection of H1N1 DNA. Specific oligonucleotide and reporter primer probes were designed together with a circular template, and the oligonucleotide probes were attached to the surfaces of magnetic beads (MBs) to form functional MB-DNA conjugates as capture probes for the target H1N1 DNA molecules. Together with the addition of DNA targets and reporter primer probes to the MB-DNA conjugates, sandwiched hybrids were formed. The initiation of RCA amplification using the circular template in the presence of phi29 polymerase allowed for the amplification of a large number of repeat sequences of the single-stranded (ss)-DNA product. This RCA product accumulated gold nanoparticles (AuNPs), resulting in a colorimetric change that could be viewed by the naked eye or detected using UV-vis spectroscopy. According to this method, H1N1 DNA could be detected at the 1 pmol L(-1) level. This platform exhibited design convenience, simplicity, and cost-effectiveness, and could be used to provide a new diagnostic assay for H1N1, and other infectious diseases.

Microfluidic Based Organ-on-Chips and Biomedical Application.

Organ-on-a-Chip is a microfluidic cell culture device manufactured via microchip fabrication methods [...].

Non-invasive detection of tumor markers in salivary extracellular vesicles based on digital PCR chips.

The World Health Organization (WHO) has stated that countless cancer patients could be saved if early detection and treatment were available. However, current clinical evaluation of tumors still relies primarily on imaging examinations and tissue biopsies. These methods not only require sophisticated equipment, but also have high false positive rates or invasive problems. Here, we describe a digital polymerase chain reaction (dPCR) chip for the detection of biomarkers in salivary extracellular vesicles (SEVs), which can be used to identify markers for the early diagnosis of tumors. Based on microfluidic technology fine microstructure and microfluidics operations, this dPCR chip can accurate quantitative SEVs in a variety of tumor markers, and shows extremely strong sensitivity (10 copies). In the detection of clinical samples, the chip can effectively distinguish lung cancer cases from normal controls (P < 0.001; two-tailed t-test), and in the detection of extremely low concentration samples, it shows considerably higher precise quantitative ability than quantitative real-time polymerase chain reaction (qPCR). Overall, this study may shed new light on non-invasive early screening of tumor markers by detecting extracellular vesicle-associated markers in saliva.

Bionic Magnetic Sensor Based on the MagR/Cry4 Complex-Configured Graphene Transistor with an Integrated On-Chip Gate.

Magnetic-sensitive proteins are regarded as key factors in animals' precise perception of the geomagnetic field. Accurate feedback on the response of these tiny proteins to magnetic fields remains a challenge. Here, we first propose a real-time accurate magnetic sensor based on the MagR/Cry4 complex-configured graphene transistor with an integrated on-chip gate. A nanometer-thick denatured bovine serum albumin film was used as the bio-interface of graphene electrolyte-gated transistors (EGTs) to immobilize the MagR/Cry4 complex. With the optimization and characterization of this bionic graphene EGT, it could detect magnetic fields in real time with a sensitivity of 1 mT, which is far lower than in earlier research. It was concluded that our MagR/Cry4 complex-configured graphene EGTs with a side-gate held great promise in terms of geomagnetic field detection. Furthermore, the constructed approach in this paper could also be utilized as a general solution for recording the response of magnetically sensitive biomolecules to magnetic fields in real time.