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.

Next-Generation Microfluidics for Biomedical Research and Healthcare Applications.

Microfluidic systems offer versatile biomedical tools and methods to enhance human convenience and health. Advances in these systems enables next-generation microfluidics that integrates automation, manipulation, and smart readout systems, as well as design and three-dimensional (3D) printing for precise production of microchannels and other microstructures rapidly and with great flexibility. These 3D-printed microfluidic platforms not only control the complex fluid behavior for various biomedical applications, but also serve as microconduits for building 3D tissue constructs-an integral component of advanced drug development, toxicity assessment, and accurate disease modeling. Furthermore, the integration of other emerging technologies, such as advanced microscopy and robotics, enables the spatiotemporal manipulation and high-throughput screening of cell physiology within precisely controlled microenvironments. Notably, the portability and high precision automation capabilities in these integrated systems facilitate rapid experimentation and data acquisition to help deepen our understanding of complex biological systems and their behaviors. While certain challenges, including material compatibility, scaling, and standardization still exist, the integration with artificial intelligence, the Internet of Things, smart materials, and miniaturization holds tremendous promise in reshaping traditional microfluidic approaches. This transformative potential, when integrated with advanced technologies, has the potential to revolutionize biomedical research and healthcare applications, ultimately benefiting human health. This review highlights the advances in the field and emphasizes the critical role of the next generation microfluidic systems in advancing biomedical research, point-of-care diagnostics, and healthcare systems.

Affinity-Based Microfluidics Combined with Atomic Force Microscopy for Isolation and Nanomechanical Characterization of Circulating Tumor Cells.

In this chapter, we present the materials and methods required to isolate and characterize circulating tumor cells (CTCs) from blood samples of cancer patients based on our newly developed microfluidic technologies. In particular, the devices presented herein are designed to be compatible with at\omic force microscopy (AFM) for post-capture nanomechanical investigation of CTCs. Microfluidics is well-established as a technology for isolating CTCs from the whole blood of cancer patients, and AFM is a gold standard for quantitative biophysical analysis of cells. However, CTCs are very scarce in nature, and those captured using standard closed-channel microfluidic chips are typically inaccessible for AFM procedures. As a result, their nanomechanical properties largely remain unexplored. Thus, given limitations associated with current microfluidic designs, significant efforts are put toward bringing innovative designs for real time characterization of CTCs. In light of this constant endeavor, the scope of this chapter is to compile our recent efforts on two microfluidic technologies, namely, the AFM-Chip and the HB-MFP, which proved to be efficient in isolating CTCs through antibody-antigen interactions, and their subsequent characterization using AFM.

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.

3D Generation of Multipurpose Atomic Force Microscopy Tips.

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.

Characterizing circulating tumor cells using affinity-based microfluidic capture and AFM-based biomechanics.

Elasticity and bio-adhesiveness of circulating tumor cells (CTCs) are important biomarkers of cancer. CTCs are rare in blood, thus their capture and atomic force microscopy (AFM)-based biomechanical characterization require use of multifunctional microfluidic device. Here, we describe procedures for fabrication of such device, AFM-Chip, and give details on its use in affinity-based CTC capture, and integration with AFM via reversable physical assembly. In the AFM-Chip, CTC capture is efficient, and transition to AFM characterization is seamless with minimal cell loss. For complete details on the use and execution of this protocol, please refer to Deliorman et al. (2020).

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.

Responses of Acinetobacter baumannii Bound and Loose Extracellular Polymeric Substances to Hyperosmotic Agents Combined with or without Tobramycin: An Atomic Force Microscopy Study.

In this work, contributions of extracellular polymeric substances (EPS) to the nanoscale mechanisms through which the multidrug-resistant Acinetobacter baumannii responds to antimicrobial and hyperosmotic treatments were investigated by atomic force microscopy. Specifically, the adhesion strengths to a control surface of silicon nitride (Si3N4) and the lengths of bacterial surface biopolymers of bound and loose EPS extracted from A. baumannii biofilms were quantified after individual or synergistic treatments with hyperosmotic agents (NaCl and maltodextrin) and an antibiotic (tobramycin). In the absence of any treatment, the loose EPS were significantly longer in length and higher in adhesion to Si3N4 than the bound EPS. When used individually, the hyperosmotic agents and tobramycin collapsed the A. baumannii bound and loose EPS. The combined treatment of maltodextrin with tobramycin collapsed only the loose EPS and did not alter the adhesion of both bound and loose EPS to Si3N4. In addition, the combined treatment was not as effective in collapsing the EPS molecules as when tobramycin was applied alone. Finally, the effects of treatments were dose-dependent. Altogether, our findings suggest that a sequential treatment could be effective in treating A. baumannii biofilms, in which a hyperosmotic agent is used first to collapse the EPS and limit the diffusion of nutrients into the biofilm, followed by the use of an antibiotic to kill the bacterial cells that escape from the biofilm because of starvation.

The relative composition of actin isoforms regulates cell surface biophysical features and cellular behaviors.

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 ß-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 ß-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 ß-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.

Role of overexpressed CFA/I fimbriae in bacterial swimming.

Enterotoxigenic Escherichia coli CFA/I is a protective antigen and has been overexpressed in bacterial vectors, such as Salmonella Typhimurium H683, to generate vaccines. Effects that overexpressed CFA/I may engender on the bacterial host remain largely unexplored. To investigate, we constructed a high CFA/I expression strain, H683-pC2, and compared it to a low CFA/I expression strain, H683-pC, and to a non-CFA/I expression strain, H683-pY. The results showed that H683-pC2 was less able to migrate into semisolid agar (0.35%) than either H683-pC or H683-pY. Bacteria that migrated showed motility halo sizes of H683-pC2 < H683-pC < H683-pY. In the liquid culture media, H683-pC2 cells precipitated to the bottom of the tube, while those of H683-pY did not. In situ imaging revealed that H683-pC2 bacilli tended to auto-agglutinate within the semisolid agar, while H683-pY bacilli did not. When the cfaBE fimbrial fiber encoding genes were deleted from pC2, the new plasmid, pC2(-), significantly recovered bacterial swimming capability. Our study highlights the negative impact of overexpressed CFA/I fimbriae on bacterial swimming motility.

Capture efficiency of Escherichia coli in fimbriae-mediated immunoimmobilization.

Capturing pathogens on a sensor surface is one of the most important steps in the design of a biosensor. The efficiency of a biosensor at capturing pathogens has direct bearing on its sensitivity. In this work we investigated the capturing of Escherichia coli on substrates modified with antibodies targeting different types of fimbriae: K88ab (F4), K88ac (F4), K99 (F5), 987P (F6), F41, and CFA/I. The results suggest that all these fimbriae can be used for the efficient immobilization of living E. coli cells. The immobilization efficiency was affected by the purity and clone type of the antibody and the fimbriae expression level of the bacteria. For a specific fimbriae type, a higher immobilization efficiency was often observed with the monoclonal antibodies. Immunoimmobilization was utilized in an antibody microarray immersed in a mixed culture of pathogens to demonstrate the rapid and simultaneous label-free detection of multiple pathogens within less than 1 h using a single test. The capture rate of living pathogens exceeds a single bacterium per 100 × 100 µm(2) area per 0.5 h of incubation for a bulk concentration of 10(5) cfu/mL.

Antibody selection for immobilizing living bacteria.

We report a comparative study of the efficacy of immobilizing living bacteria by means of seven antibodies against bacterial surface antigens associated with Salmonella enterica Serovar Typhimurium. The targeted bacterial antigens were CFA/I fimbriae, flagella, lipopolysaccharides (LPS), and capsular F1 antigen. The best immobilization of S. Typhimurium was achieved with the antibody against CFA/I fimbriae. The immobilization of bacteria using antiflagellin showed significant enhancement if the flagella rotary motion was paralyzed. Of the four antibodies targeting LPS structures, only one, the antibody against the O-antigen polysaccharides, showed a relatively efficient bacterial immobilization. No bacterial immobilization was achieved using the antibody against F1 antigen, presumably because F1 protein can detach from the bacterial surface easily. The results suggest that an antibody for bacterial immunoimmobilization should target a surface antigen which extends out from the bacterial surface and is tightly attached to the bacterial cell wall. The microarrays of living S. Typhimurium cells immobilized in this manner remained viable and effective for at least 2 weeks in growth medium before a thick biofilm covered the whole surface.

Bacteria survive multiple puncturings of their cell walls.

A bacterial cell wall is a highly dynamic multilayer structure interfacing the cytoplasm to the outside environment. It supports a multitude of chemical and biological processes necessary for life. It is therefore postulated that damage to the structure of bacterial cell wall would threaten cell integrity and result in cell death. We tested this hypothesis by repeatedly puncturing the cell wall of a live Gram negative bacterium Salmonella typhimurium at different locations using a sharp atomic force microscope nanotip and conducting multiple viability tests. Our study demonstrated that a S. typhimurium survives repeated puncturings of its cell wall and retains its integrity, viability, and ability to divide. The results are explained on the basis of the concept of the self-repairing of lipid bilayers and the peptidoglycan layer.

Porphyrin as an ideal biomarker in the search for extraterrestrial life.

A key issue in astrobiological research is identifying target molecules that are unambiguously biological in origin and can be easily detected and recognized. We suggest porphyrin derivatives as an ideal target, because these chromophores are global in distribution and found in virtually all living organisms on Earth, including microorganisms that may approximate the early evolution of life on Earth. We discuss the inherent qualities that make porphyrin ideally suited for astrobiological research and discuss methods for detecting porphyrin molecules in terrestrial sedimentary environments. We present preliminary data to support the use of ToFSIMS as a powerful technique in the identification of porphyrins.