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pH controls a large repertoire of chemical and biochemical processes in water. Densely arrayed pH microenvironments would parallelize these processes, enabling their high-throughput studies and applications. However, pH localization, let alone its arrayed realization, remains challenging because of fast diffusion of protons in water. Here, we demonstrate arrayed localizations of picoliter-scale aqueous acids, using a 256-electrochemical cell array defined on and operated by a complementary metal oxide semiconductor (CMOS)–integrated circuit. Each cell, comprising a concentric pair of cathode and anode with their current injections controlled with a sub-nanoampere resolution by the CMOS electronics, creates a local pH environment, or a pH “voxel,” via confined electrochemistry. The system also monitors the spatiotemporal pH profile across the array in real time for precision pH control. We highlight the utility of this CMOS pH localizer-imager for high-throughput tasks by parallelizing pH-gated molecular state encoding and pH-regulated enzymatic DNA elongation at any selected set of cells.

Electrode-based impedance and electrochemical measurements can provide cell-biology information that is difficult to obtain using optical-microscopy techniques. Such electrical methods are non-invasive, label-free, and continuous, eliminating the need for fluorescence reporters and overcoming optical imaging's throughput/temporal resolution limitations. Nonetheless, electrode-based techniques have not been heavily employed because devices typically contain few electrodes per well, resulting in noisy aggregate readouts. Complementary metal-oxide-semiconductor (CMOS) microelectrode arrays (MEAs) have sometimes been used for electrophysiological measurements with thousands of electrodes per well at sub-cellular pitches, but only basic impedance mappings of cell attachment have been performed outside of electrophysiology. Here, we report on new field-based impedance mapping and electrochemical mapping/patterning techniques to expand CMOS-MEA cell-biology applications. The methods enable accurate measurement of cell attachment, growth/wound healing, cell-cell adhesion, metabolic state, and redox properties with single-cell spatial resolution (20 μm electrode pitch). These measurements allow the quantification of adhesion and metabolic differences of cells expressing oncogenes

The synaptic connections between neurons are traditionally determined by correlating the action potentials (APs) of a pre-synaptic neuron and small-amplitude subthreshold potentials of a post-synaptic neuron using invasive intracellular techniques, such as patch clamping. Extracellular recording by a microelectrode array can non-invasively monitor network activities of a large number of neurons, but its reduced sensitivity usually prevents direct measurements of synaptic signals. Here, we demonstrate that a newly developed complementary metal-oxide-semiconductor (CMOS) nanoelectrode array (CNEA) is capable of extracellularly determining direct synaptic connections in dense, multi-layer cultures of dissociated rat neurons. We spatiotemporally correlate action potential signals of hundreds of active neurons, detect small (∼1 pA after averaging) extracellular synaptic signals at the region where pre-synaptic axons and post-synaptic dendrites/somas overlap, and use those signals to map synaptic connections. We use controlled stimulation to assess stimulation-dependent synaptic strengths and to titrate a synaptic blocker (CNQX: IC50 ∼ 1 μM). The new capabilities demonstrated here significantly enhance the utilities of CNEAs in connectome mapping and drug screening applications.

CMOS microelectrode arrays (MEAs) can record electrophysiological activities of a large number of neurons in parallel but only extracellularly with a low signal-to-noise ratio. Patch-clamp electrodes can perform intracellular recording with a high signal-to-noise ratio but only from a few neurons in parallel. Recently, we have developed and reported a neuroelectronic interface that combines the parallelism of the CMOS MEA and the intracellular sensitivity of the patch clamp. Here, we report the design and characterization of the CMOS integrated circuit (IC), a critical component of the neuroelectronic interface. Fabricated in 0.18- $\mu \text{m}$ technology, the IC features an array of 4096 platinum black (PtB) nanoelectrodes spaced at a 20- $\mu \text{m}$ pitch on its surface and contains 4096 active pixel circuits. Each active pixel circuit, consisting of a new switched-capacitor current injector—-capable of injecting from ±15 pA to ±0.7 $\mu \text{A}$ with a 5-pA resolution—-and an operational amplifier, is highly configurable. When configured into the current-clamp mode, the pixel intracellularly records membrane potentials, including subthreshold activities with ~23- $\mu \text{V}_{\mathrm {rms}}$ input-referred noise while injecting a current for simultaneous stimulation. When configured into the voltage-clamp mode, the pixel becomes a switched-capacitor transimpedance amplifier with ~1-pArms input-referred noise and intracellularly records ion channel currents while applying a voltage for simultaneous stimulation. Such voltage-/current-clamp intracellular recording/stimulation is a feat only previously possible with the patch-clamp method. At the same time, as an array, the IC overcomes the lack of parallelism of the patch-clamp method, measuring thousands of mammalian neurons in parallel, with full-frame intracellular recording/stimulation at 9.4 kHz.

Developing a new tool capable of high-precision electrophysiological recording of a large network of electrogenic cells has long been an outstanding challenge in neurobiology and cardiology. Here, we combine nanoscale intracellular electrodes with complementary metal-oxide-semiconductor (CMOS) integrated circuits to realize a high-fidelity all-electrical electrophysiological imager for parallel intracellular recording at the network level. Our CMOS nanoelectrode array has 1,024 recording/stimulation 'pixels' equipped with vertical nanoelectrodes, and can simultaneously record intracellular membrane potentials from hundreds of connected in vitro neonatal rat ventricular cardiomyocytes. We demonstrate that this network-level intracellular recording capability can be used to examine the effect of pharmaceuticals on the delicate dynamics of a cardiomyocyte network, thus opening up new opportunities in tissue-based pharmacological screening for cardiac and neuronal diseases as well as fundamental studies of electrogenic cells and their networks.

Ion-sensitive field-effect transistors (ISFETs) have been used to detect a variety of biomolecules whose charges alter the current or threshold voltage of the transistor. ISFETs can be built into large-scale arrays by CMOS compatible technology, offering the promise of highly parallel, all-electrical biomolecule sensing in a true chip-scale construct. Although CMOS-ISFET-based biomolecule detection has been amply demonstrated, a comprehensive optimization study of its sensitivity based on the device design, which would aid the development of large-scale arrays, remains lacking. Here, we present a systematic optimization strategy for CMOS-ISFET-based biomolecule sensing, using real-time DNA hybridization detection as an example. We analytically show that biasing the ISFETs close to the threshold is optimal, whereas minimizing the channel-to-sensing area ratio is beneficial but with limited sensitivity enhancement due to the double-layer capacitance of the sensing area. We then experimentally confirm our strategy with 26 ISFETs of varying sizes and biases from two CMOS chips, which detect the same 200-nM target DNA with different hybridization signals. The measured data correlate well to the presented theory. Our results are generally applicable to detecting other types of biomolecules, and may help in developing large-scale arrays of electrical biomolecular sensors.

The parallelization of intracellular recording can greatly benefit the study of complex neuronal networks, but it has proven difficult to achieve. To meet this challenge, we have been developing large-scale arrays of intracellular vertical nanoscale electrodes operated by underlying CMOS integrated circuits. The development has been fruitful, and our first-generation CMOS nanoelectrode array hit a milestone by intracellularly recording from up to 235 networked cardiomyocytes in parallel. We reported this first-generation system and its unprecedented parallelism in intracellular recording in Nature Nanotechnology 12, 460 (2017). The present paper is a review of this work with a special focus on the development of its CMOS integrated circuit.

Electrode technology for electrophysiology has a long history of innovation, with some decisive steps including the development of the voltage-clamp measurement technique by Hodgkin and Huxley in the 1940s and the invention of the patch clamp electrode by Neher and Sakmann in the 1970s. The high-precision intracellular recording enabled by the patch clamp electrode has since been a gold standard in studying the fundamental cellular processes underlying the electrical activities of neurons and other excitable cells. One logical next step would then be to parallelize these intracellular electrodes, since simultaneous intracellular recording from a large number of cells will benefit the study of complex neuronal networks and will increase the throughput of electrophysiological screening from basic neurobiology laboratories to the pharmaceutical industry. Patch clamp electrodes, however, are not built for parallelization; as for now, only ∼10 patch measurements in parallel are possible. It has long been envisioned that nanoscale electrodes may help meet this challenge. First, nanoscale electrodes were shown to enable intracellular access. Second, because their size scale is within the normal reach of the standard top-down fabrication, the nanoelectrodes can be scaled into a large array for parallelization. Third, such a nanoelectrode array can be monolithically integrated with complementary metal-oxide semiconductor (CMOS) electronics to facilitate the large array operation and the recording of the signals from a massive number of cells. These are some of the central ideas that have motivated the research activity into nanoelectrode electrophysiology, and these past years have seen fruitful developments. This Account aims to synthesize these findings so as to provide a useful reference. Summing up from the recent studies, we will first elucidate the morphology and associated electrical properties of the interface between a nanoelectrode and a cellular membrane, clarifying how the nanoelectrode attains intracellular access. This understanding will be translated into a circuit model for the nanobio interface, which we will then use to lay out the strategies for improving the interface. The intracellular interface of the nanoelectrode is currently inferior to that of the patch clamp electrode; reaching this benchmark will be an exciting challenge that involves optimization of electrode geometries, materials, chemical modifications, electroporation protocols, and recording/stimulation electronics, as we describe in the Account. Another important theme of this Account, beyond the optimization of the individual nanoelectrode-cell interface, is the scalability of the nanoscale electrodes. We will discuss this theme using a recent development from our groups as an example, where an array of ca. 1000 nanoelectrode pixels fabricated on a CMOS integrated circuit chip performs parallel intracellular recording from a few hundreds of cardiomyocytes, which marks a new milestone in electrophysiology.

Optical methods based on voltage sensitive proteins and Ca2+/voltage sensitive dyes have significantly benefited the field of electrophysiology, parallelizing intracellular recording from a network of electrogenic cells. Creating an analogous all-electrical device — an electrode array capable of intracellular recording from a network of electrogenic cells — has long been an outstanding challenge. On the one hand, though the CMOS microelectrode array has enabled network-level studies with massive parallelism, its extracellular interface has a limited sensitivity. On the other hand, planar patch clamp arrays achieve intracellular access but are not applicable to network-level interrogations. We will present our recent development of a CMOS nanoelectrode array that combines both parallel and intracellular features for high-precision recording across a large network of electrogenic cells, and its outlooks in neurobiology and cardiology.

Electrical sensing of biomolecules has been an important pursuit due to the label-free operation and chip-scale construct such sensing modality can enable. In particular, electrical biomolecular sensors based on nanomaterials such as semiconductor nanowires, carbon nanotubes, and graphene have demonstrated high sensitivity, which in the case of nanowires and carbon nanotubes can surpass typical optical detection sensitivity. Among these nanomaterials, graphene is well suited for a practical candidate for implementing a large-scale array of biomolecular sensors, as its two-dimensional morphology is readily compatible with industry standard top-down fabrication techniques. In our recent work published in 2014 Nature Communications, we demonstrated these benefits by creating DNA sensor arrays from chemical vapor deposition (CVD) graphene. The present chapter, which is a review of this recent work, outlines procedures demonstrating the use of individual graphene sites of the array in dual roles--electrophoretic electrodes for site specific probe DNA immobilization and field effect transistor (FET) sensors for detection of target DNA hybridization. The 100 fM detection sensitivity achieved in 7 out of 8 graphene FET sensors in the array combined with the alternative use of the graphene channels as electrophoretic electrodes for probe deposition represent steps toward creating an all-electrical multiplexed DNA array.

Electrical sensing of biomolecules has been an important pursuit due to the label-free operation and chip-scale construct such sensing modality can enable. In particular, electrical biomolecular sensors based on nanomaterials such as semiconductor nanowires, carbon nanotubes, and graphene have demonstrated high sensitivity, which in the case of nanowires and carbon nanotubes can surpass typical optical detection sensitivity. Among these nanomaterials, graphene is well suited for a practical candidate for implementing a large-scale array of biomolecular sensors, as its two-dimensional morphology is readily compatible with industry standard top-down fabrication techniques. In our recent work published in 2014 Nature Communications, we demonstrated these benefits by creating DNA sensor arrays from chemical vapor deposition (CVD) graphene. The present chapter, which is a review of this recent work, outlines procedures demonstrating the use of individual graphene sites of the array in dual roles--electrophoretic electrodes for site specific probe DNA immobilization and field effect transistor (FET) sensors for detection of target DNA hybridization. The 100 fM detection sensitivity achieved in 7 out of 8 graphene FET sensors in the array combined with the alternative use of the graphene channels as electrophoretic electrodes for probe deposition represent steps toward creating an all-electrical multiplexed DNA array.

The continuing global burden of disease from influenza infections reveals an urgent need for new approaches that more efficiently protect and potentially avoid the emergence of drug-resistant strains over time. One strategy could be to therapeutically target natural killer T (NKT) cells, an innate-like lymphocyte subset capable of inducing potent antiviral immune responses. We previously reported that disease outcome is significantly improved when influenza-infected pigs are intranasally treated with the NKT cell superagonist α-galactosylceramide (αGC). Here, we compared the protective effects of αGC to 7DW8-5, an αGC derivative that promotes pro-inflammatory immune responses, as well as oseltamivir phosphate (Tamiflu®), a neuraminidase inhibitor used to treat and prevent influenza. A total of 20 pigs challenged with H1N1 A/California/04/2009 (CA04) (4 per group) were administered vehicle, 7DW8-5, or αGC and Tamiflu®, alone or in combination. Four other pigs were mock infected and vehicle treated. Symptoms, viral shedding and peripheral blood leukocytes were measured for 7 days post infection (dpi). Pigs were then euthanized to examine viral titers and immune responses in the airway and lymphoid organs. Pigs treated with αGC and 7DW8-5 but not Tamiflu restored weight gain of infected pigs to the level of un-infected animals. All treatments eliminated virus shedding by 2 dpi compared to infected and untreated pigs that continued to shed virus throughout the study. So far, our results suggest that NKT cell therapy is similar to Tamiflu® for reducing the severity and transmission of influenza infections in pigs. Thus, it may be possible to use NKT cell superagonists to limit the spread of influenza viruses amongst swine and humans.

Type 1 diabetes (T1D) is an autoimmune disease in which insulin-producing pancreatic β cells are destroyed by auto-aggressive T cells. Tregs in T1D have been shown to lose their suppressive function and/or become inflammatory, complicating Treg immunotherapy. Oral treatment with the adhesin from enterotoxigenic E. coli, CFA/I fimbriae, protects against autoimmunity. Although CFA/I fimbriae act in a bystander or in an antigen (Ag)-independent fashion, protection is ultimately dependent upon the induction and/or activation of auto-Ag-specific Tregs. Our initial findings showed that oral dosing with a Lactococcus lactis vector expressing CFA/I fimbriae (LL-CFA/I) reduces incidence of T1D in NOD mice by 45%. We hypothesized that dosing NOD mice more frequently would induce Foxp3+ Tregs in the pancreas and PaLNs to provide more robust protection. To test this, 4 wk-old NOD mice were orally dosed with LL-CFA/I varying the dosage and frequency of treatment. When dosed every 2 wks, the frequency of insulitis was reduced by more than half at 11 wks of age, and tetramer staining revealed a 2-fold reduction of insulin-specific CD4+ and CD8+ T cells within the PaLNs. Additional groups were followed until 16 wks of age, and examined for Treg and inflammatory T cell phenotypes. Treatment with LL-CFA/I reduced 2-fold IFN-γ-producing CD8+ T cells in the spleen and PaLNs. PaLN Foxp3+ T cells from the PBS and vector control groups, but not the LL-CFA/I-treated mice, co-expressed Tbet and IFN-γ, suggesting that Tregs from LL-CFA/I treated mice maintained their regulatory function at 16 wks. These data show that orally dosing with LL-CFA/I diminishes or halts T1D progression via activation of stably functional Foxp3+ Tregs. Work supported by AI121745.

Oral treatment with the adhesin from enterotoxigenic E. coli, CFA/I fimbriae protect in murine models of multiple sclerosis and rheumatoid arthritis. Although CFA/I fimbriae initial mode of action is in a bystander or in an antigen (Ag)-independent fashion, protection was found to be ultimately dependent upon the induction and/or activation of auto-Ag-specific regulatory T cells (Tregs). Our recent findings show that oral dosing with a L. lactis vector expressing CFA/I fimbriae (LL-CFA/I) reduces incidence of T1D in NOD mice by 45% with a concomitant 8-fold increase in their splenic Foxp3+CD25+ Tregs. However, little is known about how protection transitions from bystander suppression to Ag-specific Tregs. We hypothesized that LL-CFA/I stimulates dendritic cells (DCs) to establish a regulatory microenvironment since DCs play an integral role in fate decisions for T cells becoming inflammatory or tolerogenic. In this study, 4 wk-old NOD mice were orally dosed with LL-CFA/I and treated every 2 wks; control groups were given L. lactis vector or PBS. At 11 wks of age, frequency of insulitis was reduced by more than half, and insulin-specific T cells were reduced in the pancreatic lymph nodes (PaLNs; P < 0.05). To discern how this was mediated, groups of mice were examined at 3, 7, 14, and 21 days post-treatment for changes in DC and T cell phenotypes. As early as 3 days post-treatment, DCs exhibited significantly less expression of costimulatory molecules, CD40 and CD86, and stably maintained this phenotype for at least 7 wks. Splenic DCs showed reduced IL-6 production. These data show that orally dosing with LL-CFA/I ameliorates T1D in NOD mice by establishing a regulatory microenvironment via DCs. Work is supported by AI121745.

Field-effect transistor biomolecular sensors based on low-dimensional nanomaterials boast sensitivity, label-free operation and chip-scale construction. Chemical vapour deposition graphene is especially well suited for multiplexed electronic DNA array applications, since its large two-dimensional morphology readily lends itself to top-down fabrication of transistor arrays. Nonetheless, graphene field-effect transistor DNA sensors have been studied mainly at single-device level. Here we create, from chemical vapour deposition graphene, field-effect transistor arrays with two features representing steps towards multiplexed DNA arrays. First, a robust array yield--seven out of eight transistors--is achieved with a 100-fM sensitivity, on par with optical DNA microarrays and at least 10 times higher than prior chemical vapour deposition graphene transistor DNA sensors. Second, each graphene acts as an electrophoretic electrode for site-specific probe DNA immobilization, and performs subsequent site-specific detection of target DNA as a field-effect transistor. The use of graphene as both electrode and transistor suggests a path towards all-electrical multiplexed graphene DNA arrays.

A 6-year-old castrated dromedary camel (Camelus dromedarius) presented with a non-healing, severely pruritic, ulcerative fibrotic plaque located at the medial canthus. Histological examination of surgical biopsies identified degenerating nematode larvae within eosinophilic granulomas. Treatment involved repeated debridement of the lesion, injectable ivermectin and anti-inflammatory therapies, and injectable and topical antibiotics. A specially constructed mask with goggles to prevent the camel from continuing to self-traumatize the eye and lesion was also placed. Full recovery occurred approximately 1 month after diagnosis. Because of the location of the lesion, time of year, the gross and microscopic characteristics of the lesion, the presence of a likely nematode larva and the response to treatment, a diagnosis of cutaneous habronemiasis was made.

CMOS technology and its Moore’s Law scaling is an enormously successful technology paradigm that has continued to transform our computation and communication abilities. Outside the applications in computation and communication, CMOS technology has been increasingly applied to the life sciences, with a wealth of silicon integrated circuits developed to interface with biological molecules and cells. Concretely, large-scale arrays of active electrodes are integrated using CMOS technology for highly parallel electronic detection of biomolecular/ionic charges and cellular potentials for DNA sequencing, molecular diagnostics, and electrophysiology. Parallelism enabled by CMOS scalability is well suited to process the big data in these biotechnological applications. Here we offer a brief review on these CMOS-bio interfaces, while the corresponding presentation will focus on a sub-topic of CMOS electrophysiology with mammalian neurons.

Session: Semantic Multimedia (short paper); International audience; Execution traces have become essential resources that many developers analyze to debug their applications. Ideally, a developer wants to quickly detect whether there are anomalies on his application or not. However, in practice, the size of multimedia applications trace can reach gigabytes, which makes their exploitation very complex. Usually, developers use visualization tools before stating a hypothesis. In this paper, we argue that this solution is not satisfactory and propose to automatically provide a diagnosis by comparing execution traces. We use distance-based models and conduct a user case to show how TED, our automatic trace diagnosis tool, provides semantic added-value information to the developer. Performance evaluation over real world data shows that our approach is scalable.