OMEF biochip for evaluating red blood cell deformability using dielectrophoresis as a diagnostic tool for type 2 diabetes mellitus.

Type 2 diabetes mellitus (T2DM) is a prevalent and debilitating disease with numerous health risks, including cardiovascular diseases, kidney dysfunction, and nerve damage. One important aspect of T2DM is its association with the abnormal morphology of red blood cells (RBCs), which leads to increased blood viscosity and impaired blood flow. Therefore, evaluating the mechanical properties of RBCs is crucial for understanding the role of T2DM in cellular deformability. This provides valuable insights into disease progression and potential diagnostic applications. In this study, we developed an open micro-electro-fluidic (OMEF) biochip technology based on dielectrophoresis (DEP) to assess the deformability of RBCs in T2DM. The biochip facilitates high-throughput single-cell RBC stretching experiments, enabling quantitative measurements of the cell size, strain, stretch factor, and post-stretching relaxation time. Our results confirm the significant impact of T2DM on the deformability of RBCs. Compared to their healthy counterparts, diabetic RBCs exhibit ∼27% increased size and ∼29% reduced stretch factor, suggesting potential biomarkers for monitoring T2DM. The observed dynamic behaviors emphasize the contrast between the mechanical characteristics, where healthy RBCs demonstrate notable elasticity and diabetic RBCs exhibit plastic behavior. These differences highlight the significance of mechanical characteristics in understanding the implications for RBCs in T2DM. With its ∼90% sensitivity and rapid readout (ultimately within a few minutes), the OMEF biochip holds potential as an effective point-of-care diagnostic tool for evaluating the deformability of RBCs in individuals with T2DM and tracking disease progression.

Spike- and nucleocapsid-based gold colloid assay toward the development of an adhesive bandage for rapid SARS-CoV-2 immune response detection and screening.

Immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies are important biomarkers used for the diagnosis and screening of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections in both symptomatic and asymptomatic individuals. These antibodies are highly specific to the spike (S) and nucleocapsid (N) proteins of the SARS-CoV-2 virus. This paper outlines the development steps of a novel hybrid (vertical-lateral-vertical) flow assay in the form of a finger-stick point-of-care device, similar to an adhesive bandage, designed for the timely detection and screening of IgM and IgG immune responses to SARS-CoV-2 infections. The assay, comprising a vertically stacked plasma/serum separation membrane, conjugate pad, and detection (readout) zone, utilizes gold nanoparticles (AuNPs) conjugated with SARS-CoV-2 S and N proteins to effectively capture IgM and IgG antibodies from a pinprick (~15 µL) of blood in just one step and provides results of no immune IgM-/IgG-, early immune IgM+/IgG-, active immune IgM+/IgG+ or immune IgM-/IgG+ in a short amount of time (minutes). The adhesive bandage-like construction is an example of the design of rapid, low-cost, disposable, and easy-to-use tests for large-scale detection and screening in households. Furthermore, the bandage can be easily adjusted and optimized to detect different viral infections as they arise by simply selecting appropriate antigens related to pandemics and outbreaks.

Noncontact Multiphysics Probe for Spatiotemporal Resolved Single-Cell Manipulation and Analyses.

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.

Cryopreservable arrays of paper-based 3D tumor models for high throughput drug screening.

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.

Airplug-mediated isolation and centralization of single T cells in rectangular microwells for biosensing.

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 µm3) 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.

Paper-Based Cell Cryopreservation.

The continuous development of simple and practical cell cryopreservation methods is of great importance to a variety of sectors, especially when considering the efficient short- and long-term storage of cells and their transportation. Although the overall success of such methods has been increased in recent years, there is still need for a unified platform that is highly suitable for efficient cryogenic storage of cells in addition to their easy-to-manage retrieval. Here, a paper-based cell cryopreservation method as an alternative to conventional cryopreservation methods is presented. The method is space-saving, cost-effective, simple and easy to manage, and requires no additional fine-tuning to conventional freezing and thawing procedures to yield comparable recovery of viable cells. It is shown that treating papers with fibronectin solution enhances the release of viable cells post thawing as compared to untreated paper platforms. Additionally, upon release, the remaining cells within the paper lead to the formation and growth of spheroid-like structures. Moreover, it is demonstrated that the developed method works with paper-based 3D cultures, where preformed 3D cultures can be efficiently cryopreserved.

AFM-compatible microfluidic platform for affinity-based capture and nanomechanical characterization of circulating tumor cells.

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.

A Method to Efficiently Cryopreserve Mammalian Cells on Paper Platforms.

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.

Selective and vertical microfabrication of lipid tubule arrays on glass substrates using template-guided gentle hydration.

Generally, asymmetric tubular lipid structures have been formed under the specific condition of gentle hydration or by using hydrodynamic and/or electrical elongation of vesicular lipid structures. Small-size lipid tubes are, however, very difficult to allocate or align in the vertical direction on the specific site of the substrate and, therefore, the ability to produce them selectively and in large quantities as an array form is limited. Herein, we propose an easy and novel method to fabricate selective and vertical lipid tube arrays using template-guided gentle hydration of dried lipid films without any external forces. A lipid solution was drop-dispensed onto a porous membrane and dried to form a lipid film. Then, the lipid-coated porous membrane was transferred to a glass substrate by using a UV-cured polymer layer to achieve tight bonding. Upon swelling with an appropriate buffer, expansion forces due to osmotic pressure during the gentle hydration process were highly constrained to confined pores, thereby resulting in the nucleation of tube-like lipid structures through the pores. Interestingly, according to the aspect ratio of pores (ARpore, pore length/pore diameter), different shapes of lipid structures, including vesicular, oval, and tube-like, were generated, which indicates the importance of the ARpore, as well as the pore diameter, during fabrication of tubular lipid structures. Also, this approach was easily modified with 1% chitosan to enhance the stability of the lipid tubes (>30 min in life time), by lipid coating twice and by using unsaturated lipids to increase tube length (>30 µm in length). Therefore, in the future, the simple but robust template-guided gentle hydration method will be a useful tool for fabricating addressable and engineered lipid tube arrays as a sensory unit.