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In this work, 3D polymeric atomic force microscopy (AFM) tips, referred to as 3DTIPs, are manufactured with great flexibility in design and function using two-photon polymerization. With the technology holding a great potential in developing next-generation AFM tips, 3DTIPs prove effective in obtaining high-resolution and high-speed AFM images in air and liquid environments, using common AFM modes. In particular, it is shown that the 3DTIPs provide high-resolution imaging due to their extremely low Hamaker constant, high speed scanning rates due to their low quality factor, and high durability due to their soft nature and minimal isotropic tip wear; the three important features for advancing AFM studies. It is also shown that refining the tip end of the 3DTIPs by focused ion beam etching and by carbon nanotube inclusion substantially extends their functionality in high-resolution AFM imaging, reaching angstrom scales. Altogether, the multifunctional capabilities of 3DTIPs can bring next-generation AFM tips to routine and advanced AFM applications, and expand the fields of high speed AFM imaging and biological force measurements.

We have developed a rapid technique for characterizing the biomechanical properties of dendritic cells using dielectrophoretic forces. It is widely recognized that maturing of dendritic cells modulates their stiffness and migration capabilities, which results in T-cell activation triggering the adaptive immune response. Therefore it is important to develop techniques for mechanophenotyping of immature and mature dendritic cells. The technique reported here utilizes nonuniform electric fields to exert a substantial force on the cells to induce cellular elongation for optical measurements. In addition, a large array of interdigitated electrodes allows multiple cells to be stretched simultaneously. Our results indicate a direct correlation between F-actin activity and deformability observed in dendritic cells, determined through mean fluorescence signal intensity of phalloidin.

We present an electrically actuated approach for creating a well-defined centered microparticle cluster within a sessile droplet on an interdigitated microelectrodes. The method is demonstrated with different aggregation shapes and particle types including biological cells for 3D microtissue development. AC voltage application induces particle levitation and enhanced-convection through accelerated evaporation. Radial long-range fluid convection evolves along the substrate surface toward the droplet’s center and suspended microparticles aggregate within the central stagnation zone, in an interesting occurrence that is opposite to the well-known coffee ring effect. This remarkable approach could open new opportunities in immunoassays, rare cell counting, and 3D cell cultures.

Summary Cellular microenvironments are dynamic. When exposed to extracellular cues, such as changing concentrations of inflammatory cytokines, cells activate signaling networks that mediate fate decisions. Exploring responses broadly to time-varying microenvironments is essential to understand the information transmission capabilities of signaling networks and how dynamic milieus influence cell fate decisions. Here, we present a gravity-driven cell culture and demonstrate that the system accurately produces user-defined concentration profiles for one or more dynamic stimuli. As proof of principle, we monitor nuclear factor-κB activation in single cells exposed to dynamic cytokine stimulation and reveal context-dependent sensitivity and uncharacterized single-cell response classes distinct from persistent stimulation. Using computational modeling, we find that cell-to-cell variability in feedback rates within the signaling network contributes to different response classes. Models are validated using inhibitors to predictably modulate response classes in live cells exposed to dynamic stimuli. These hidden capabilities, uncovered through dynamic stimulation, provide opportunities to discover and manipulate signaling mechanisms., Graphical Abstract, Highlights • Automated and accessible analog control of dynamic cell culture microenvironments • Gravity-driven flow enables long-term modulation for multiple time-varying stimuli • Atypical patterns of NF-κB activation are revealed by dynamic stimulation with TNF • Protein translation rate is a source of variability between NF-κB response classes, Immunology; Mathematical Biosciences; Complex Systems; Biological Science Instrumentation

Introduction : A single-lead electrocardiogram (EKG) is routinely recorded with electroencephalogram (EEG). This study investigates the frequency and types of EKG abnormalities during routine EEG. Methods : All routine EEGs (20–60 min) over one year were retrospectively analyzed. A blinded cardiologist interpreted EKG recordings. An epileptologist evaluated EEGs. Demographic data, underlying comorbidities, and indications for the EEG were extracted. Results : A total of 433 recordings for 365 patients were included. Mean (±SD) age was 46.8 (±21.3) years and 50.4% were females. EKG abnormalities were detected in 28.5% of patients; sinus tachycardia (11%), premature ventricular contractions (7.9%), atrial fibrillation (Afib) (6.3%), sinus bradycardia (2.2%) and premature atrial contractions (1.1%). Afib was more common in females than males (p = 0.020), confirmed in six out of seven patients and discovered in 17 patients. Age (OR: 1.67, 95%CI: 1.05–2.66, p = 0.031), prior diagnosis of epilepsy (OR: 2.25, 95% CI: 1.22 – 4.14, p = 0.009), history of seizure (OR: 1.97, 95%CI: 1.09–3.54, p = 0.024), abnormal EEG (OR: 2.14, 95%CI: 1.25 – 3.66, p = 0.005) and EEGs evaluating seizures/epilepsy (OR: 4.18, 95% CI: 1.32 – 13.21, p = 0.015) or syncope (OR: 3.21, 95% CI: 1.16 – 8.84, p = 0.024) were independently associated with abnormal EKG. Conclusion : The frequency of EKG abnormalities captured during routine EEGs was high, with Afib being the most significant. Older age, history of epilepsy or seizure, abnormal EEGs, and EEGs evaluating seizures/epilepsy or syncope were significant predictors. These findings suggest neurologists to become more vigilant to EKG recorded during routine EEG as such findings might have diagnostic and therapeutic implications.

Three-dimensional (3D) tumor models have gained increased attention in life-science applications as they better represent physiological conditions of in vivo tumor microenvironments, and thus, possess big potential for guiding drug screening studies. Although various techniques proved effective in growing cancer cells in 3D, their procedures are typically complex, time consuming, and expensive. Here, we present a versatile, robust, and cost-effective method that utilizes a paper platform to create cryopreservable high throughput arrays of 3D tumor models. In the approach, we use custom 3D printed masks along with simple chemistry modifications to engineer highly localized hydrophilic ‘virtual microwells’, or microspots, on paper for 3D cell aggregation, surrounded by hydrophobic barriers that prevent inter-microspot mixing. The method supports the formation and cryopreservation of 3D tumor arrays for extended periods of storage time. Using MCF-7 and MDA-MB-231 breast cancer cell lines, we show that the cryopreservable arrays of paper-based 3D models are effective in studying tumor response to cisplatin drug treatment, while replicating key characteristics of the in vivo tumors that are absent in conventional 2D cultures. This technology offers a low cost, easy, and fast experimental procedure, and allows for 3D tumor arrays to be cryopreserved and thawed for on-demand use. This could potentially provide unparalleled advantages to the fields of tissue engineering and personalized medicine.

Sorting cells in a single cell per microwell format is of great interest to basic biology studies, biotherapeutics, and biosensing including cell phenotyping. For instance, isolation of individual immune T cells in rectangular microwells has been shown to empower the multiplex cytokine profiling at the single cell level for therapeutics applications. The present study, however, shows that there is an existing bias in temporal cytokine sensing that originates from random “unpredicted” positions of loaded cells within the rectangular microwells. To eliminate this bias, the isolated cells need to be well-aligned with each other and relative to the sensing elements. Hence, an approach that utilizes the in situ formation and release of airplugs to localize cells towards the center of the rectangular microwells is reported. The chip includes 2250 microwells (each 500 × 50 × 20 μm(3)) arranged in 9 rows. Results showed 20% efficiency in trapping single T cells per microwells, where cells are localized within ±3% of the center of microwells. The developed platform could provide real-time dynamic and unbiased multiplex cytokine detection from single T cells for phenotyping and biotherapeutics studies.

This protocol describes a simple method to cryopreserve mammalian cells within filter papers as an alternative to conventional slow-freezing approach. The method involves treating paper fibers with fibronectin, using low concentrations of the cryoprotectant dimethyl sulfoxide (DMSO), and slow freezing cells to -80 °C at a 1 °C min(-1) rate. In our method, the biocompatibility, large surface area, 3D porosity and fiber flexibility of the paper, in combination with the fibronectin treatment, yield recovery of cells comparable to conventional approaches, with no additional fine-tuning to freezing and thawing procedures. We expect that the paper-based cryopreservation method will bring several advantages to the field of preserving mammalian cells, including accommodation of a higher number of cells within a unit volume and no cell loss after release. The method requires a minimal storage space, where paper platforms with large areas can be rolled and/or folded and stored in stocks, and allows for efficient transportation/distribution of cells in an on-demand manner. Moreover, an additional feature of this method includes the formation and cryopreservation of cellular spheroids and 3D cell cultures.

Circulating tumor cells (CTCs) are attracting increasing interest in medical sciences as they play a key role to understanding cancer metastasis. The possibility to phenotype CTCs based on their physiological nature, biological responses, and defense mechanisms could open new venues for precision diagnosis, personalized treatments, and preventative measures. In this work, we employed Atomic Force Microscopy (AFM) for mechanophenotyping clinical prostate CTCs based on their measured elastic and adhesive properties. We performed the AFM force measurements on different cancer cell lines, namely MCF7 cells (breast cancer), LNCaP and PC3 cells (prostate cancer), and compared the results with clinical CTCs isolated from blood of prostate cancer patients.

In this work, we develop microfluidic probes (MFPs) by utilizing high resolution 3D printing, and demonstrate applications in targeted drug delivery with a single cell resolution. In addition, the MFP’s features are extended to include heating elements in order to facilitate its application as a single cell micro-thermo-fluidic tweezer – a heat assisted microfluidic single cell tweezer. With the high resolution 2photon photolithography 3D printing technique (Nanoscribe), we achieve microfluidic injection apertures of $30 \mu \mathrm{m}$ diameter which corresponds to the average span of single cells in adherent culture. Flow characterization demonstrates the devices’ capability of rapidly adjusting its localized target area from about 100 cells (32$,000 \mu \mathrm{m}^{2}$) to single cell resolutions $(320 \mu \mathrm{m}^{2}$). Results show that these resolutions can be maintained in the scanning mode to achieve high resolution drug delivery in cultured adherent cells. Furthermore, as an advancement to the conventional MFP, pin shape electrodes were introduced at the tip to incorporate localized heating features for optimal cell dissociation conditions. Characterization of the heat generated by the pin electrodes is reported and an application of the micro-thermo-fluidic tweezer to selectively detach and collect single adherent cells is demonstrated.

Background Autoimmune encephalitis presents with subacute neurological, psychiatric, and behavioral symptoms. In many cases, MRI and CSF studies return normal. Laboratory testing for pathogenic neuronal antibodies is limited by accessibility and sensitivity issues. Finally, the different diagnostic approaches lack consensus and have until recently been largely retrospective. Collectively, these factors predispose for delays in diagnosis and treatment in many cases. Case presentation We describe the case of a 42-year-old woman who did not initially meet the newly proposed criteria for probable autoimmune encephalitis, but in whom the detection on electroencephalogram (EEG) of subtle asymmetric slowing of the left hemisphere ultimately led to the diagnosis. Discussion EEG is highly sensitive and rarely normal in encephalitis. The recently introduced diagnostic criteria for autoimmune encephalitis included new-onset seizures in the earliest steps in the algorithm but did not yet incorporate EEG findings. We encourage practicing physicians to use EEG as an early, sensitive diagnostic tool for cases suspected to have autoimmune encephalitis.

Circulating tumor cells (CTCs) carried by the patient’s bloodstream are known to lead to the metastatic spread of cancer. It is becoming increasingly clear that an understanding of the nanomechanical characteristics of CTCs, such as elasticity and adhesiveness, represents advancements in tracking and monitoring cancer progression and metastasis. In the present work, we describe a combined microfluidic–atomic force microscopy (AFM) platform that uses antibody–antigen capture to routinely isolate and nanomechanically characterize CTCs present in blood samples from prostate cancer patients. We introduce the reversible assembly of a microfluidic device and apply refined and robust chemistry to covalently bond antibodies onto its glass substrate with high density and the desired orientation. As a result, we show that the device can efficiently capture CTCs from patients with localized and metastatic prostate cancer through anti-EpCAM, anti-PSA, and anti-PSMA antibodies, and it is suitable for AFM measurements of captured intact CTCs. When nanomechanically characterized, CTCs originating from metastatic cancer demonstrate decreased elasticity and increased deformability compared to those originating from localized cancer. While the average adhesion of CTCs to the AFM tip surface remained the same in both the groups, there were fewer multiple adhesion events in metastatic CTCs than there were in their counterparts. The developed platform is simple, robust, and reliable and can be useful in the diagnosis and prognosis of prostate cancer as well as other forms of cancer.

Background Cell surface mechanics is able to physically and biomechanically affect cell shape and motility, vesicle trafficking and actin dynamics. The biophysical properties of cell surface are strongly influenced by cytoskeletal elements. In mammals, tissue-specific expression of six actin isoforms is thought to confer differential biomechanical properties. However, the relative contribution of actin isoforms to cell surface properties is not well understood. Here, we sought to investigate whether and how the composition of endogenous actin isoforms directly affects the biomechanical features of cell surface and cellular behavior. Methods We used fibroblasts isolated from wild type (WT), heterozygous (HET) and from knockout (KO) mouse embryos where both β-actin alleles are not functional. We applied a combination of genome-wide analysis and biophysical methods such as RNA-seq and atomic force microscopy. Results We found that endogenous β-actin levels are essential in controlling cell surface stiffness and pull-off force, which was not compensated by the up-regulation of other actin isoforms. The variations of surface biophysical features and actin contents were associated with distinct cell behaviors in 2D and 3D WT, HET and KO cell cultures. Since β-actin in WT cells and smooth muscle α-actin up-regulated in KO cells showed different organization patterns, our data support the differential localization and organization as a mechanism to regulate the biophysical properties of cell surface by actin isoforms. Conclusions We propose that variations in actin isoforms composition impact on the biophysical features of cell surface and cause the changes in cell behavior.

Stem cell research has great potential in clinical applications and regenerative medicine, which highlight the need for efficient stem cell isolation technologies. Enrichment of stem cells is challenging because of its sensitivity to the surrounding environment and its overlapping features with non-target cells. Current gold-standard isolation techniques depend on surface antigens that are exclusively expressed on stem cells, where surface immuno-labeling is followed by flow cytometry or magnetic sorting procedures. It is well-established that stem cells should be enriched with minimal alteration, therefore, isolation without immunological tagging in a label-free format is of great interest. Microfluidic technologies have demonstrated high level of control to precisely manipulate stem cells. Here, we review the recent emerging label-free microfluidic based cell isolation technologies that provide unbiased stem cell enrichment based on their size and deformability, adhesion, and electrical characteristics, and discuss their potential to achieve effective stem cell isolation for research and clinical applications.

The microfluidic probe (MFP) is a noncontact technology that applies the concept of hydrodynamic flow confinement (HFC) within a small gap to eliminate the need for closed microfluidic conduits and, therefore, overcome the conventional closed-system microfluidic limitation. Since its invention, the concept has experienced continuing advancement with several applications, ranging from manipulating mammalian cells and printing protein arrays to performing microfabrication.

Cardiovascular is a disorder of the heart and its blood vessels that causes millions of deaths around the world. Implanting stent in patient’s artery is a typical procedure of treatment. However, a major problem associated with stents is the buildup of plaque/fatty acids deposits, a condition known as in-stent restenosis. Therefore, one of the preventive solutions is the development of "smart" stents that contain pressure sensors for monitoring stent blockage at early stages. Such sensors need to be associated with high sensitivity for early detection of flow abnormalities within the stent. Here, we propose several capacitive pressure sensor plate designs and analyze their sensitivities. Our results show that a square-shaped plate with straight slots on its edges can drastically improve sensitivity. Several analyses were carried out to optimize dimensions of the proposed design for monitoring in-stent restenosis.

Cell separation and patterning are of interest to several biological and medical applications including rare cell isolation and co-culture models. Numerous microfluidic devices have been used for cell separation and patterning, however, the typical closed channel configuration comes with challenges and limitations. Here, we report a dielectrophoresis (DEP) enabled microelectrofluidic probe (MeFP) for sequentially separating and patterning of mammalian cells in an open microfluidic system. The MeFP is a microfluidic probe with injection and aspiration apertures, integrated with an array of micro-hump electrodes on its tip. Aligning the MeFP parallel, and in close proximity, to a conductive substrate forms a vertical pin-plate electrode configuration that allows for an integration of DEP forces within the hydrodynamic flow confinement. Upon confining a heterogeneous cell suspension in the gap between the MeFP and the substrate, target cells are selectively captured on the micro-hump electrodes using positive DEP forces, and then deposited on the substrate in defined patterns. Characterization of the MeFP showed an increase in cell-capture efficiency when the MeFP is of a higher microfluidic multipole configuration. Separation of cancer cells from T lymphocytes was demonstrated with capture purity as high as 89.6%. Deposited patterns of isolated cells match the numerically calculated particle trajectories of the evaluated microfluidic multipoles configurations. By adjusting the flow configuration of the MeFP, we show that the patterned co-culture of two different cell types can be dynamically controlled for homotypic and heterotypic cell interaction studies. This work presents a multifunctional microfluidic tool that bio-fabricates selective multicellular patterns directly on an open substrate without the need for confined conduits.

We have developed a rapid and novel technique for concentrating cells into 3D clusters which are then embedded in an agar hydrogel. This method supports cell growth and proliferation with a steady nutrient supply through the porous hydrogel. For tissue engineering studies, the creation of cell clusters within a controlled size range is necessary to perform precise biological experiments and measure functions. Our precisely controlled cell clusters can be reproducibly formed for different sizes between 0.1 mm to 5 mm in diameter, with cell numbers up to 10 6 cells/cluster. The concentration of cells is achieved using a device we previously developed, that utilizes a combination of electrical and hydrodynamic phenomena. The results were obtained using an 80 μL drop containing HeLa cancer cells placed over an interdigitated electrode array. The electrohydrodynamic phenomena was produced using a sinusoidal voltage (10 V pk-pk and 50 kHz). This voltage application creates Joule heating resulting in a buoyancy driven convective flow pattern directed towards the center of the droplet. In addition, the same interdigitated electrode structure also induces dielectrophoretic (DEP) levitation of the suspended cells which prevents cell settling and non-specific adhesion. The dense clustering of cells is achieved within 10 minutes of AC voltage application. Furthermore, using staining we have demonstrated that cell viability was measured as 90% after the formation of agar-embedded cell clusters and 80% over 72 hours.

Effective capture of cancer cells from whole peripheral blood samples, i.e. circulating tumor cells (CTCs), is still an existing limitation for liquid biopsy-based diagnostics. The well-established closed-channel herringbone micro-mixers are one of the widely adopted methods for isolating CTCs based on antigen-antibody interaction. However, they are known to be associated with several drawbacks, such as limited capture areas within the channels, restricted access to isolated cells, difficulties to achieve multiplexed antibody capture assays for immuno-phenotyping, and limited postprocessing possibilities. To tackle these issues, we developed a novel microfluidic probe (MFP) that is integrated with herringbone micro-mixers on its tip surface (HMFP). The tip surface was designed with 2-slitted apertures, one for injecting the cell suspension and the other for performing high flow rate aspiration to confine the flow. The herringbone mixing elements were distributed in-between the apertures for micro-mixing that enhances the CTCs capture on the antibodies-coated bottom glass surface. Unlike the closed herringbone chips, the functionalized bottom glass surface was kept large given the capacity for the MFP to work in scanning mode, and so it prevented cell capture saturation effect. Our MFP design and experimental setup showed a cell capture efficiency of 59-81% with flow rates of 0.6-2.4 mL/h. The capture of CTCs in an open microfluidic system allows for easy post-process and CTC analysis, such as single cell drug testing and mechano-phenotyping using atomic force microscopy.

This work presents the development of a micro-electro-fluidic probe (MeFP) platform as an affordable and flexible microfluidic tool for the transfection of single cells via electroporation. The platform constitutes of a 3D printed MeFP -- gold-coated microfluidic probe (MFP) with an array of pin shaped microelectrodes integrated on its tip -- and an ITO coated cell culture substrate. This setup, and submicron feature size of the MeFP, allows for a selective exposure of the targeted cell to both the electric field and hydrodynamic flow confinement (HFC) of an intercalating agent, to demonstrate transmembrane molecule delivery through electroporation. Results show successful transfer of propidium iodide (PI) through the membranes of single HeLa cells with an applied DC rectangular pulse– a proof-of-concept for MeFP’s application in delivering nucleic acids into eukaryotic cells (transfection). By adjusting the size of the HFC (varying injection and aspiration flow ratio), we show that the cell target area can be dynamically increased from the single cell footprint, to cover multiple cells. Finite Element model show that even with such low applied voltages (0.5- 3Vpk-pk), the electric field generated reach the reversible electroporation threshold. These results demonstrate the MeFP as an advancement to the currently available transfection technologies for gene therapy; delivery of DNA vaccines, in vitro fertilization, cancer treatment, regenerative medicine, and induced pluripotent stem (iPS) cells.

Here, we numerically justify precise positioning of single cells in rectangular microwells for accurate secretory protein sensing, and report a microfluidic single cell per microwell positioning device. Our results show significant time and concentration bias in multiplexed cytokine sensors based on the position of the single cells relative to the sensor. By combining air plugs with immunochemistry in a microfluidic chip, single cells were successfully positioned at the center of 450 microwells. This device could potentially play a vital role in acquiring precise sensitivity and specificity in secretory protein temporal sensing at the single cell level.

Heterogeneity and spatial arrangement of individual cells within tissues are critical to the identity of the host multicellular organism. While current single-cell techniques are capable of resolving heterogeneity, they mostly rely on extracting target cells from their physiological environment and hence lose the spatiotemporal resolution required for understanding cellular networks. Here, a multifunctional noncontact scanning probe that can precisely perform multiple manipulation procedures on living single-cells, while within their physiological tissue environment, is demonstrated. The noncontact multiphysics probe (NMP) consists of fluidic apertures and "hump" shaped electrodes that simultaneously confine reagents and electric signals with a single-cell resolution. The NMP's unique electropermealization-based approach in transferring macromolecules through the cell membrane is presented. The technology's adjustable spatial ability is demonstrated by transfecting adjacent single-cells with different DNA plasmid vectors. The NMP technology also opens the door for controllable cytoplasm extraction from living single-cells. This powerful application is demonstrated by executing multiple time point biopsies on adherent cells without affecting the integrity of the extracted macromolecules or the viability of cells. Furthermore, the NMP's function as an electro-thermal based microfluidic whole-cell tweezer is reported. This work offers a multifunctional tool with unprecedented probing features for spatiotemporal single-cell analysis within tissue samples.

Isolated microfluidic stagnation point flows – stagnation point flows formed within microfluidic interfaces – have come a long way as a tool for characterizing materials, trapping/manipulating micro particles, and generating confined flows and localized chemistries. Early applications of these fluidic stagnation flows focused on characterizing emulsions and polymers. However, in recent times, these flows have taken microfluidic forms to expand the range of applications to single-cell analysis, substrate patterning, and chip-integrated devices, amongst others. This article focuses on reviewing the microfluidics literature to highlight the concept behind isolated microfluidic stagnation point flows, underline different approaches of experimentally resolving them, and then look into their applications in chemistry and life sciences; with a specific focus on micro-total-analysis systems. The literature on this topic is approached historically; from the isolated fluidic stagnation point flow generated by the early four-roll mill to the currently predominating microfluidic stagnation point flows of the cross-slot device. Finally, isolated microfluidic stagnation flows produced by the relatively recent open space microfluidics – opposed jets, microfluidic probes and micropipettes – are critically analyzed and their applications are discussed. Ultimately, the goal of this article is to inform the scientific community on the constructive aspect of microfluidic stagnation flows by demonstrating their past, present, and future applicability in analytical chemistry and life sciences.