An Integrated Droplet Manipulation Platform with Photodeformable Microfluidic Channels.

Manipulating droplets by light in microscale allows precise control of microfluidics, liquid delivery, micromachines, and so on. Among these applications, microfluidic technology is of particular interest for miniaturization of the portable analysis systems, which require the integration of various liquid operations in one device. Here, a photodeformable microfluidic platform is constructed by combining Laplace pressure and capillary condensation to integrate the transportation, fusion, separation, and mixing of liquid slugs in one chip. The Laplace pressure, attributed to the photodeformation of the liquid crystal polymers, is generated to propel the slug. The capillary condensation is introduced by the delicate design of the fluid channels, allowing the fusion and separation of slugs without any connected microvalves. Catalytic oxidation reaction and protein detection processes are realized in the platform, which are amenable to a variety of miniaturized bio-medical applications, such as portable analysis and point of care testing.

A quantum dot microspheres-based highly specific and sensitive three-dimensional microarray for multiplexed detection of inflammatory factors.

The development trend ofin vitrodiagnostics is to obtain various biological information from a sample at extremely low concentration and volume, which has promoted its progress in accurate and sensitive multiplexed detection. Here, we developed a single color quantum dot (QD) based three-dimensional (3D) structure matrix microarray and conducted the detection of two inflammatory factors (C-reactive protein (CRP) and serum amyloid A (SAA)) by a self-built fluorescence detection system. This strategy increased detection sensitivity by immobilizing the antibody specifically on the 3D substrate because it captured more than about 7 times of 'effective' antibodies compared to the two-dimensional (2D) plane. Compared to the dual QDs-2D fluorescence-linked immunosorbent assay, the limit of detection (LOD) of 3D microarray based on QDs modified with amphiphilic polymers has been further improved to 0.11 ng ml-1for SAA assay and to 0.16 ng ml-1for CRP assay, respectively. By using QD microspheres (SiO2@QDs@SiO2-COOH, containing approximately 200-300 hydrophobic QDs on per SiO2sphere) as fluorescent labels, the LOD for CRP and SAA of 3D microarray reached as high as 15 pg ml-1and 86 pg ml-1, and the sensitivity was further improved by 28-fold and 425-fold, respectively. Because of its excellent performance, this QD microspheres-based 3D microarray has great application potential for highly sensitive and multiplexed quantitative detection of other biomarkers, small molecules, and antibiotic residues in biomedicine and food safety.

Human neuronal networks on micro-electrode arrays are a highly robust tool to study disease-specific genotype-phenotype correlations in vitro.

Micro-electrode arrays (MEAs) are increasingly used to characterize neuronal network activity of human induced pluripotent stem cell (hiPSC)-derived neurons. Despite their gain in popularity, MEA recordings from hiPSC-derived neuronal networks are not always used to their full potential in respect to experimental design, execution, and data analysis. Therefore, we benchmarked the robustness of MEA-derived neuronal activity patterns from ten healthy individual control lines, and uncover comparable network phenotypes. To achieve standardization, we provide recommendations on experimental design and analysis. With such standardization, MEAs can be used as a reliable platform to distinguish (disease-specific) network phenotypes. In conclusion, we show that MEAs are a powerful and robust tool to uncover functional neuronal network phenotypes from hiPSC-derived neuronal networks, and provide an important resource to advance the hiPSC field toward the use of MEAs for disease phenotyping and drug discovery.

Acoustic Droplet-Assisted Superhydrophilic-Superhydrophobic Microarray Platform for High-Throughput Screening of Patient-Derived Tumor Spheroids.

Cell-based high-throughput screening is a key step in the current disease-based research, drug development, and precision medicine. However, it is challenging to establish a rapid culture and screening platform for rare cells (patient-derived) due to the obvious differences between the traditional 2D cell model and the tumor microenvironment, as well as the lack of a low-consumption screening platform for low numbers of cells. Here, we developed an acoustic drop-assisted superhydrophilic-superhydrophobic microarray platform for the rapid culture and screening of a few cells. By employing hydrophilic and hydrophobic microarrays, we can automatically distribute the cell suspension into uniform droplets, and these cells can spontaneously form compact 3D cell spheroids within 36 h (similar to the microenvironment of tumors in vivo). By using the acoustic droplet ejection device, we can accurately inject a drug solution with a volume of ∼pL to ∼nL into the droplet, and the whole process can be completed within 20 ms (one print). By using three different cell lines (Caco-2, MCF-7, and HeLa) to optimize the platform, the culture and screening of five patients' colon cancer were subsequently realized. Using three conventional chemotherapeutics (5-fluorouracil, cetuximab, and panitumumab) of various concentrations, the best treatment was screened out and compared with the actual treatment effect of the patients, and the results were extremely similar. As a proof-of-concept application, we have proved that our platform can quickly cultivate patient samples and effectively screen the best treatment methods, highlighting its wide application in precision medicine, basic tumor research, and drug development.

Multifunctional Printable Micropattern Array for Digital Nucleic Acid Assay for Microbial Pathogen Detection.

The digital nucleic acid assay is a precise, sensitive, and reproducible method for determining the presence of individual target molecules separated in designated partitions; thus, this technique can be used for the nucleic acid detection. Here, we propose a multifunctional micropattern array capable of isolating individual target molecules into partitions and simultaneous on-site cell lysis to achieve a direct DNA extraction and digitized quantification thereof. The multifunctional micropattern array is fabricated by the deposition of a copolymer film, poly(2-dimethylaminomethyl styrene-co-hydroxyethyl methacrylate) (pDH), directly on a microfluidic chip surface via the photoinitiated chemical vapor deposition process, followed by hydrophobic microcontact printing (µCP) to define each partition for the nucleic acid isolation. The pDH layer is a positively charged surface, which is desirable for the bacterial lysis and DNA capture, while showing exceptional water stability for more than 24 h. The hydrophobic µCP-treated pDH surface is stable under aqueous conditions at a high temperature (70 °C) for 1 h and enables the rapid and reliable formation of thousands of sessile microdroplets for the compartmentalization of an aqueous sample solution without involving bulky and costly microfluidic devices. By assembling the multifunctional micropattern array into the microfluidic chip, the isothermal amplification in each partition can detect DNA templates over a concentration range of 0.01-2 ng/µL. The untreated bacterial cells can also be directly compartmentalized via the microdroplet formation, followed by the on-site cell lysis and DNA capture on the compartmentalized pDH surface. For Escherichia coli O157:H7, Salmonella enteritidis, and Staphylococcus aureus cells, cell numbers ranging from 1.4 × 104 to 1.4 × 107 can be distinguished by using the multifunctional micropattern array, regardless of the cell type. The multifunctional micropattern array developed in this study provides a novel multifunctional compartmentalization method for rapid, simple, and accurate digital nucleic acid assays.

Tunable Nanopore Arrays as the Basis for Ionic Circuits.

There has been considerable interest in preparing ionic circuits capable of manipulating ionic and molecular transport in a solution. This direction of research is inspired by biological systems where multiple pores with different functionalities embedded in a cell membrane transmit external signals and underlie all physiological processes. In this manuscript, we describe the modeling of ion transport through small arrays of nanopores consisting of 3, 6, and 9 nanopores and an integrated gate electrode placed on the membrane surface next to one pore opening. We show that by tuning the gate voltage and strategically placing nanopores with nonlinear current-voltage characteristics, the local signal at the gate affects ionic transport through all nanopores in the array. Conditions were identified when the same gate voltage induced opposite rectification properties of neighboring nanopores. We also demonstrate that an ionic diode embedded in a nanopore array can modulate transport properties of neighboring pores even without a gate voltage. The results are explained by the role of concentration polarization and overlapping depletion zones on one side of the membrane. The modeling presented here is intended to become an inspiration to future experiments to create nanopore arrays that can transduce signals in space and time.

Integrating modification and detection in acoustic microchip for in-situ analysis.

Ultrasound as a biocompatible and powerful approach has been advanced in biotechnology. Here we present an acoustic microchip integrating modification and detection for in-situ analysis. Such microchip employs two pairs of piezoelectric transducers (PZTs) for acoustic field generation and a polydimethylsiloxane (PDMS) microcavity on a polyethylene terephthalate (PET) substrate for producing microparticle array. The applying of acoustic field results in rapidly forming microparticle array by adjusting the inputting frequency and voltage. In-situ modification and detection are accelerated due to the dynamic ultrasonic streaming around the ultrasound induced microparticle array. Such array also benefits from reducing the detection errors by coupling of multiple points. With this strategy, biomarkers (e.g. miRNA) can be enriched, and achieve in-situ modification and detection via simple two steps with excellent specificity. After the detection, samples are regained from the output channel by releasing the acoustic field, which is benefit for further analysis. Such integrated modification and detection acoustic microchip shows great potential in visual in-situ analysis and enriching ultratrace biomarkers for clinical diagnosis.

Non-enzymatic electrochemical determination of creatinine using a novel screen-printed microcell.

A low-cost and disposable microcell was constructed with a screen-printed electrode for the non-enzymatic electrochemical determination of creatinine. The working electrode was modified with carbon black and maintained in contact with paper-adsorbed iron (III) ions. A small sample volume of 3 µL was required for the device operation. Then, iron (III) ions were complexed in the presence of creatinine in a chemical step, followed by an electrochemical reduction of non-complexed metallic ions in excess. Cyclic voltammetry and differential-pulse voltammetry experiments were employed for the electrochemical characterizations and analytical performance evaluation of the microcell. The working electrode modification with carbon black provided a significant increase of analytical signal. The sensor presented a linear response for creatinine concentrations ranging from 0.10 to 6.5 mmol L-1, with a limit of detection of 0.043 mmol L-1. Experiments for creatinine determination in real samples were successful performed through of standard recovery in urine.

Stochastic deposition of amino acids into microcavities via microparticles.

All known methods for solid-phase synthesis of molecular arrays exploit positioning techniques to deposit monomers on a substrate preferably high densely. In this paper, stochastic patterning of molecule spots (250 000 spots monomers/cm2) via random allocation of the microbeads on a microstructured glass is presented. The size and shape of the microbeads and the microcavities are selected in such a way so that only one microbead can fit into the respective microcavity. Each microbead can be loaded with a certain type of molecule e.g. amino acids and is brought in the microcavities stochastically. Applying solvent vapor and heating the substrate, the molecules are released from the microbeads and coupled to the functionalized substrate. To differentiate between the microbeads carrying different molecules, quantum dot labels are preliminary introduced into the microbeads. Fluorescence imaging and subsequent data analysis enable decoding of the molecule deposition patterns. After the coupling step is completed, the microbeads are mechanically removed from the microwells. The composition of the monomer microbeads, their deposition and the conditions of the monomer extraction are studied. The stochastic monomer patterning may be used to design novel molecular arrays.

A multichannel magnetic stimulation system using submillimeter-sized coils: system development and experimental application to rodent brain in vivo.

OBJECTIVE: Single coil-based systems for magnetic stimulation are widely used for neurostimulation in neuroscience research and clinical treatment of neurological diseases. However, parallelization of magnetic stimulation with multiple coils may generate far greater potential than a single coil, and could thus expand the scope of brain area stimulation. Therefore, we examined whether a multiple coil-based system could improve the effectiveness and focality of conventional single coil-based magnetic stimulation. APPROACH: We designed and tested a micromagnetic stimulation (µMS) device with multiple submillimeter-sized coils as a possible substitute for one large coil. Our design concept is spatially-distributed stimulation strategy involving the small number of coils to be able to mimic desired electric field profiles. To this end, the cost function of the error between the desired and coil-induced electric fields was firstly calculated, and coil currents were repetitively estimated to achieve the smaller number of coils under a certain criterion: a minimum error with spatial sparsity. Using these approaches, we evaluated the capability of our multi-channel µMS via numerical simulations and demonstrated responsive results in animal experiments. MAIN RESULTS: Our approach can enhance control of neural excitation and improve the concentration of the excitation field induced by magnetic stimulation with reduced power consumption. Furthermore, in vivo electrophysiological recordings of mouse brain performed to evaluate our proposed approach for brain stimulation demonstrated experimentally that our multi-channel µMS device can yield more effective stimulation than the single-channel device. In addition, our device permitted electronic spatial adjustment of the stimulus shape and location without moving the coils. SIGNIFICANCE: The development of new multichannel µMS-based therapeutic approaches may be useful because the µMS affects only a restricted brain area. Indeed, the small size of micro-coils and their finer focality with multichannel contribution might be suitable for chronic use, which is difficult using conventional large transcranial magnetic stimulation (TMS) with simple round or figure-eight coils. Thus, our findings support new opportunities to explore magnetic stimulation as a therapeutic approach for neurological disorders.

Large 10 × 10 single cell grid networks of human hNT astrocytes on raised parylene-C/SiO2 substrates.

OBJECTIVE: The 'Astrocytic Network' is an emerging research field for researchers in cell biology. Culturing astrocytes in organised networks is a novel method for permitting controlled studies and investigations into the calcium transients of such networks. Recent research has photolithographically patterned hNT astrocytes on parylene-C inlayed SiO2 trench grid networks. However, it was observed that the trench networks could not specifically immobilise the astrocyte cell bodies to the nodes of the networks. APPROACH: In this study, for the first time, we demonstrate how it is possible to establish grid networks of human hNT astrocytes on raised parylene-C structures where the cell bodies are specifically organised down to the single-cell level on nodes of the grid and connected throughout. MAIN RESULTS: Here, we report these to be the largest patterned single-cell grid network of astrocytes of their kind consisting of 100 cells in a 10 × 10 grid arrangement to an 80% efficiency. We quantify the level of patterning through six cell patterning assessment indices: the parylene adhesion index (PAI); SiO2 attraction index (SAI); node index (NI) and connectivity interval (χI), number of components (k) and fielder value (λ ss) and report that the best connected network is obtained with 65 µm node size, 90 µm node spacing, and 5 µm interconnecting track width (PAI = 0.77 ± 0.040, SAI = 0.12 ± 0.049, NI = 0.81 ± 0.066, χI = 0.25 ± 0.064, k = 2.33 ± 1.528, λ ss = 0.0249 ± 0.0018). We finally demonstrate, through delivery of ATP, that the networks are functional on the raised parylene-C grid structures. SIGNIFICANCE: The significance of this study is that it determines the optimal dimensions to obtain highly organised, large, interlinked, single-cell networks which provide an effective platform to investigate calcium communication within astrocytic networks in an accurate, controlled and repeatable manner.

Ultrasimple Single-Cell Detection of Multiple Cytokines by a Nanowell Chip Integrated with Encoded Microarrays.

Cytokine production is often regarded as the marker of immune cells' activation status. The spectrum and temporal secretion of cytokines are dramatically varied between cell phenotypes and even within the same phenotype. Multiparameter analysis of individual immune cell's cytokine secretion has always been a challenging and complicated process that needs special facilities in a laboratory setting. Herein, we present an ultrasimple method with high sensitivity and high robustness to quantify cytokine expression at the single-cell resolution. A microchip is developed based on poly(dimethylsiloxane) nanowells on sticky tape, while each nanowell is integrated with a DNA-antibody convertible microarray. Only pipetting is needed for the whole single-cell analysis process. The sensitivity of the assay is evaluated by measuring various concentrations of six recombinant cytokine proteins, which was found comparable to conventional methods. Once single cells are loaded to nanowells and incubated there, a Fluorinert FC-40 is used to isolate nanowells; so, cytokines from those cells are captured by separate microarrays. The rest of the sandwich enzyme-linked immunosorbent assay detection process is also executed simply by pipetting of various reagents. This method is validated by measuring cytokine production from hundreds of single cells. It has simplified a typically sophisticated multiplex single-cell assay into an instrument-free, point-of-detection technology, and thus it may find a broad utility in clinical diagnostics.

Hierarchically structured microchip for point-of-care immunoassays with dynamic detection ranges.

Point-of-care (POC) medical assays provide critical information to guide clinical therapy for a broad range of medical scenarios, such as resource-poor settings and specialty departments in hospitals. Even though many types of POC assays can be done in automated devices, these POC assays typically cannot well accommodate the multiplexed detection of biomarkers where a large dynamic range is needed. Here, we report a POC assay, which is both automated and suitable for detecting multiple biomarkers with dynamic detection ranges. We call it a dynamic multiplexed immunoassay (DMI). We control the concentrations of capture antibodies and the intensity of the readout signal to dynamically modulate the detection range of immunoassays (pg mL-1 to µg mL-1), leading to the multiplexed detection of C-reactive protein (CRP), procalcitonin (PCT), and interleukin 6 (IL-6) simultaneously in undiluted human serum samples. The POC assay allows the rapid and accurate detection of infection in patients.

Improving the SERS signals of biomolecules using a stacked biochip containing Fe2O3/Au nanoparticles and a DC magnetic field.

This study proposes a magnetic biochip that uses surface-enhanced Raman scattering (SERS) for antigen detection. The biochip was a sandwich structure containing alternating layers of gold and magnetic Fe2O3 nanoparticles. Both single (Au/Fe2O3/Au) and multilayer (Au/Fe2O3/Au/Fe2O3/Au) chips containing Fe2O3 nanoparticles were fabricated to detect bovine serum albumin (BSA). The single-layer chip detected the BSA antigen at a signal-to-noise ratio (SNR) of 5.0. Peaks detected between 1000 and 1500 cm-1 corresponded to various carbon chains. With more Fe2O3 layers, bond resonance was enhanced via the Hall effect. The distribution of electromagnetic field enhancement was determined via SERS. The signal from the single-layer chip containing Au nanoparticles was measured in an external magnetic field. Maximum signal strength was recorded in a field strength of 12.5 gauss. We observed peaks due to other carbon-hydrogen molecules in a 62.5-gauss field. The magnetic field could improve the resolution and selectivity of sample observations.

Early sepsis diagnosis via protein and miRNA biomarkers using a novel point-of-care photonic biosensor.

Sepsis is a condition characterized by a severe stage of blood-infection often leading to tissue damage, organ failure and finally death. Fast diagnosis and identification of the sepsis stage (sepsis, severe sepsis or septic shock) is critical for the patient's evolution and could help in defining the most adequate treatment in order to reduce its mortality. The combined detection of several biomarkers in a timely, specific and simultaneous way could ensure a more accurate diagnosis. We have designed a new optical point-of-care (POC) device based on a phase-sensitive interferometric biosensor with a label-free microarray configuration for potential high-throughput evaluation of specific sepsis biomarkers. The sensor chip, which relies on the use of metallic nanostructures, provides versatility in terms of biofunctionalization, allowing the efficient immobilization of different kind of receptors such as antibodies or oligonucleotides. We have focused on two structurally different types of biomarkers: proteins, including C-reactive protein (CRP) and Interleukin 6 (IL6), and miRNAs, using miRNA-16 as an example. Limits of Detection (LoD) of 18 µg mL-1, 88 µg mL-1 and 1 µM (6 µg mL-1) have been respectively obtained for CRP, IL6 and miRNA-16 in individual assays, with high accuracy and reproducibility. The multiplexing capabilities have also been assessed with the simultaneous analysis of both protein biomarkers.

A Photochromic Sensor Microchip for High-Performance Multiplex Metal Ion Detection.

Photochromic molecules can respond to external stimulations and undergo reversible conversion between different chemical structures, providing one photochromic molecule with multiple recognition states for targeting compounds. Here we design a facile sensor microchip with only one photochromic molecule (spirooxazine) to discriminate multiplex metal ions. The sensor chip performs in dark, ultraviolet, or visual stimulation, resulting in different molecular states of spirooxazine-metallic coordination and patterned fluorescent signals for analysis. By using this sensor microchip, 11 metal ions are discriminated. Furthermore, mineral water of 16 different brands and metal ions in human serum are distinguished.

Encoded Microneedle Arrays for Detection of Skin Interstitial Fluid Biomarkers.

Skin interstitial fluid (ISF) is considered as an emerging source of biomarkers with physiological and medical significance. Microneedle arrays (MNs) provide a promising means for painless, noninvasive detection of these biomarkers. Here, novel MNs integrated with photonic crystal (PhC) barcodes are presented, and multiplex specific detection of ISF biomarkers is realized for the first time. The PhC barcodes-loaded flexible MNs are simply fabricated by replicating dynamic ferrofluid-cast micromoldings. When the prepared MNs are inserted into skin, they can enrich specific biomarkers to their probes-decorated PhC barcodes. Thus, by adding corresponding fluorescent probes to form sandwich immunocomplexes, the relative content of the biomarkers can be read out through the fluorescence intensity of the barcodes; meanwhile, the species of these biomarkers can be clearly distinguished by the reflection peaks of the PhC barcodes. Based on the encoded MNs, their sensitivity, flexibility, and versatility of capturing and detecting three inflammatory cytokines are demonstrated in a sepsis mice model. Compared with existing MNs for ISF detection, the encoded MNs not only possess equivalent detection effects with less post-processing and simplified procedures, but can also detect multiple biomarkers simultaneously, which makes them ideal in many clinical and biomedical detection areas.

Multivalent glycan arrays.

Glycan microarrays have become a powerful technology to study biological processes, such as cell-cell interaction, inflammation, and infections. Yet, several challenges, especially in multivalent display, remain. In this introductory lecture we discuss the state-of-the-art glycan microarray technology, with emphasis on novel approaches to access collections of pure glycans and their immobilization on surfaces. Future directions to mimic the natural glycan presentation on an array format, as well as in situ generation of combinatorial glycan collections, are discussed.

A novel synthetic-genetic-array-based yeast one-hybrid system for high discovery rate and short processing time.

Eukaryotic gene expression is often tightly regulated by interactions between transcription factors (TFs) and their DNA cis targets. Yeast one-hybrid (Y1H) is one of the most extensively used methods to discover these interactions. We developed a high-throughput meiosis-directed yeast one-hybrid system using the Magic Markers of the synthetic genetic array analysis. The system has a transcription factor-DNA interaction discovery rate twice as high as the conventional diploid-mating approach and a processing time nearly one-tenth of the haploid-transformation method. The system also offers the highest accuracy in identifying TF-DNA interactions that can be authenticated in vivo by chromatin immunoprecipitation. With these unique features, this meiosis-directed Y1H system is particularly suited for constructing novel and comprehensive genome-scale gene regulatory networks for various organisms.