Dissolvable Calcium Alginate Microfibers Produced via Immersed Microfluidic Spinning.

Fabrication of micro- and nanofibers are critical for a wide range of applications from microelectronics to biotechnology. Alginate microfibers with diameters of tens to hundreds of microns play an important role in tissue engineering and fibers of these diameters are impossible to fabricate via electrospinning and can only be produced via fluidic spinning. Typically, microfluidic spinning based on photopolymerization produces fibers that are not easily dissolvable, while fluidic spinning with chemical cross-linking employs complex setups of microfabricated chips or coaxial needles, aimed at precise control of the fiber diameter; however, fluidic spinning introduces significant cost and complexity to the microfluidic setup. We demonstrate immersed microfluidic spinning where a calcium alginate microfiber is produced via displacement of alginate solution through a single needle that is immersed in a cross-linking bath of calcium chloride solution. The resulting diameter of the fiber is characterized and the fiber diameter and topology of the deposited fiber is related to the concentration of the alginate solution (2 wt%, 4 wt%, and 6 wt%), needle gauge (30 g, 25 g, and 20 g), and the volumetric flow rate of the alginate solution (1 mL/min, 2 mL/min, and 2.7 mL/min). The resulting fiber diameter is smaller than the internal diameter of the needle and this dependence is explained by the continuity of the flow and increased rate of fall of the liquid jet upon its issuing from the needle. The fiber diameter (demonstrated diameter of fibers range from 100 microns to 1 mm) depends weakly on the volumetric flow rate and depends strongly on the needle diameter. It also seems that for a smaller needle size, a greater concentration of alginate results in smaller diameter fibers and that this trend is not evident as the needle diameter is increased. In terms of topology of the deposited fiber, the higher wt% alginate fiber produces larger loops, while smaller wt% alginate solution yields a denser topology of the overlaid fiber loops. These fibers can be dissolved in DMEM/EDTA/DSC solution in 20-30 min (depending on the fiber diameter), leaving behind the hollow channels in the hydrogel matrix. We believe that the demonstrated simple setup of the immersed microfluidic spinning of the calcium alginate microfibers will be useful for creating tissue constructs, including the vascularized tissue implants.

The immune response to COVID-19: Does sex matter?

The coronavirus disease 2019 (COVID-19) pandemic has created unprecedented challenges worldwide. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19 and has a complex interaction with the immune system, including growing evidence of sex-specific differences in the immune response. Sex-disaggregated analyses of epidemiological data indicate that males experience more severe symptoms and suffer higher mortality from COVID-19 than females. Many behavioural risk factors and biological factors may contribute to the different immune response. This review examines the immune response to SARS-CoV-2 infection in the context of sex, with emphasis on potential biological mechanisms explaining differences in clinical outcomes. Understanding sex differences in the pathophysiology of SARS-CoV-2 infection will help promote the development of specific strategies to manage the disease.

A Machine Learning-Assisted Nanoparticle-Printed Biochip for Real-Time Single Cancer Cell Analysis.

Cancers are a complex conglomerate of heterogeneous cell populations with varying genotypes and phenotypes. The intercellular heterogeneity within the same tumor and intratumor heterogeneity within various tumors are the leading causes of resistance to cancer therapies and varied outcomes in different patients. Therefore, performing single-cell analysis is essential to identify and classify cancer cell types and study cellular heterogeneity. Here, the development of a machine learning-assisted nanoparticle-printed biochip for single-cell analysis is reported. The biochip is integrated by combining powerful machine learning techniques with easily accessible inkjet printing and microfluidics technology. The biochip is easily prototype-able, miniaturized, and cost-effective, potentially capable of differentiating a variety of cell types in a label-free manner. n-feature classifiers are established and their performance metrics are evaluated. The biochip's utility to discriminate noncancerous cells from cancerous cells at the single-cell level is demonstrated. The biochip's utility in classifying cancer sub-type cells is also demonstrated. It is envisioned that such a chip has potential applications in single-cell studies, tumor heterogeneity studies, and perhaps in point-of-care cancer diagnostics-especially in developing countries where the cost, limited infrastructures, and limited access to medical technologies are of the utmost importance.

3D-bioprinted all-inclusive bioanalytical platforms for cell studies.

Innovative drug screening platforms should improve the discovery of novel and personalized cancer treatment. Common models such as animals and 2D cell cultures lack the proper recapitulation of organ structure and environment. Thus, a new generation of platforms must consist of cell models that accurately mimic the cells' microenvironment, along with flexibly prototyped cell handling structures that represent the human environment. Here, we adapted the 3D-bioprinting technology to develop multiple all-inclusive high throughputs and customized organ-on-a-chip-like platforms along with printed 3D-cell structures. Such platforms are potentially capable of performing 3D cell model analysis and cell-therapeutic response studies. We illustrated spherical and rectangular geometries of bio-printed 3D human colon cancer cell constructs. We also demonstrated the utility of directly 3D-bioprinting and rapidly prototyping of PDMS-based microfluidic cell handling arrays in different geometries. Besides, we successfully monitored the post-viability of the 3D-cell constructs for seven days. Furthermore, to mimic the human environment more closely, we integrated a 3D-bioprinted perfused drug screening microfluidics platform. Platform's channels subject cell constructs to physiological fluid flow, while its concave well array hold and perfused 3D-cell constructs. The bio-applicability of PDMS-based arrays was also demonstrated by performing cancer cell-therapeutic response studies.

A Low-Cost, Disposable and Portable Inkjet-Printed Biochip for the Developing World.

Electrowetting on dielectric-based digital microfluidic platforms (EWOD-DMF) have a potential to impact point-of-care diagnostics. Conventionally, EWOD-DMF platforms are manufactured in cleanrooms by expert technicians using costly and time consuming micro-nanofabrication processes such as optical lithography, depositions and etching. However, such high-end microfabrication facilities are extremely challenging to establish in resource-poor and low-income countries, due to their high capital investment and operating costs. This makes the fabrication of EWOD-DMF platforms extremely challenging in low-income countries, where such platforms are most needed for many applications such as point-of-care testing applications. To address this challenge, we present a low-cost and simple fabrication procedure for EWOD-DMF electrode arrays, which can be performed anywhere with a commercial office inkjet printer without the need of expensive cleanroom facilities. We demonstrate the utility of our platform to move and mix droplets of different reagents and physiologically conductive buffers, thereby showing its capability to potentially perform a variety of biochemical assays. By combining our low-cost, inkjet-printed EWOD-DMF platform with smartphone imaging technology and a compact control system for droplet manipulation, we also demonstrate a portable and hand-held device which can be programmed to potentially perform a variety of biochemical assays.

Microtechnology-based methods for organoid models.

Innovations in biomaterials and stem cell technology have allowed for the emergence of novel three-dimensional (3D) tissue-like structures known as organoids and spheroids. As a result, compared to conventional 2D cell culture and animal models, these complex 3D structures have improved the accuracy and facilitated in vitro investigations of human diseases, human development, and personalized medical treatment. Due to the rapid progress of this field, numerous spheroid and organoid production methodologies have been published. However, many of the current spheroid and organoid production techniques are limited by complexity, throughput, and reproducibility. Microfabricated and microscale platforms (e.g., microfluidics and microprinting) have shown promise to address some of the current limitations in both organoid and spheroid generation. Microfabricated and microfluidic devices have been shown to improve nutrient delivery and exchange and have allowed for the arrayed production of size-controlled culture areas that yield more uniform organoids and spheroids for a higher throughput at a lower cost. In this review, we discuss the most recent production methods, challenges currently faced in organoid and spheroid production, and microfabricated and microfluidic applications for improving spheroid and organoid generation. Specifically, we focus on how microfabrication methods and devices such as lithography, microcontact printing, and microfluidic delivery systems can advance organoid and spheroid applications in medicine.

Microinjectrode System for Combined Drug Infusion and Electrophysiology.

This microinjectrode system is designed for drug infusion, electrophysiology, and delivery and retrieval of experimental probes, such as microelectrodes and nanosensors, optimized for repeated use in awake, behaving animals. The microinjectrode system can be configured for multiple purposes: (1) simple arrangement of the cannula for placement of an experimental probe that would otherwise be too fragile to penetrate the dura mater, (2) microfluidic infusion of a drug, either independently or coupled to a cannula containing an experimental probe (i.e., microelectrode, nanosensor). In this protocol we explain the step by step construction of the microinjectrode, its coupling to microfluidic components, and the protocol for use of the system in vivo. The microfluidic components of this system allow for delivery of volumes on the nanoliter scale, with minimal penetration damage. Drug infusion can be performed independently or simultaneously with experimental probes such as microelectrodes or nanosensors in an awake, behaving animal. Applications of this system range from measuring the effects of a drug on cortical electrical activity and behavior, to understanding the function of a specific region of cortex in the context of behavioral performance based on probe or nanosensor measurements. To demonstrate some of the capabilities of this system, we present an example of muscimol infusion for reversible inactivation of the frontal eye field (FEF) in rhesus macaque during a working memory task.

Personalized Drug Efficacy Monitoring Chip.

Cancer drug resistance mechanisms such as tumor heterogeneity and adaptable feedback loops are prevalent issues facing cancer therapy development. Drug resistance can be unique to a cancer type and, most importantly, to each individual cancer patient. Consequently, testing different dosages and therapeutics directly on each individual patient sample (i.e., tumor and cancer cells) has compelling advantages compared to large scale in vitro drug testing and is a step toward personalized drug selection and effective treatment development. Recently, microfluidic-based chemo-sensitivity assays on patient biopsies have been proposed. Despite their novelty, these platforms usually rely on optical labels, optical equipment, or complex microfabricated channel geometries and structures. In this work, we proposed a novel lab on a chip platform capable of real-time and continuous screening of drug efficacy on (cancer) cell subpopulations without the need of labels or bulky readout optical equipment. In this platform, several label-free and rapid techniques have been implemented for the precise capturing of cells of interest in parallel with the real-time measurement and characterization of the effectiveness of candidate therapeutic agents. To demonstrate the utility of the platform, the effect of an apoptotic inducer, topoisomerase I inhibitor, 7-ethyl-10-hydrocamptothecin (SN38) on human colorectal carcinoma cancer cells (HCT 116) was used as a study model. Additionally, electrical results were optically verified to examine the continuous measurements of the biological mechanisms, specifically, apoptosis and necrosis, during therapeutic agent characterizations. The proposed device is a versatile platform which can also be easily redesigned for the automated and arrayed analysis of cell-drug interaction down to the single cell level. Our platform is another step toward enabling the personalized screening of drug efficacy on individual patients' samples that potentially leads to a better understanding of drug resistance and the optimization of patients' treatments.

Multifunctional, inexpensive, and reusable nanoparticle-printed biochip for cell manipulation and diagnosis.

Isolation and characterization of rare cells and molecules from a heterogeneous population is of critical importance in diagnosis of common lethal diseases such as malaria, tuberculosis, HIV, and cancer. For the developing world, point-of-care (POC) diagnostics design must account for limited funds, modest public health infrastructure, and low power availability. To address these challenges, here we integrate microfluidics, electronics, and inkjet printing to build an ultra-low-cost, rapid, and miniaturized lab-on-a-chip (LOC) platform. This platform can perform label-free and rapid single-cell capture, efficient cellular manipulation, rare-cell isolation, selective analytical separation of biological species, sorting, concentration, positioning, enumeration, and characterization. The miniaturized format allows for small sample and reagent volumes. By keeping the electronics separate from microfluidic chips, the former can be reused and device lifetime is extended. Perhaps most notably, the device manufacturing is significantly less expensive, time-consuming, and complex than traditional LOC platforms, requiring only an inkjet printer rather than skilled personnel and clean-room facilities. Production only takes 20 min (vs. up to weeks) and $0.01-an unprecedented cost in clinical diagnostics. The platform works based on intrinsic physical characteristics of biomolecules (e.g., size and polarizability). We demonstrate biomedical applications and verify cell viability in our platform, whose multiplexing and integration of numerous steps and external analyses enhance its application in the clinic, including by nonspecialists. Through its massive cost reduction and usability we anticipate that our platform will enable greater access to diagnostic facilities in developed countries as well as POC diagnostics in resource-poor and developing countries.

Nanoelectronic three-dimensional (3D) nanotip sensing array for real-time, sensitive, label-free sequence specific detection of nucleic acids.

The improvements in our ability to sequence and genotype DNA have opened up numerous avenues in the understanding of human biology and medicine with various applications, especially in medical diagnostics. But the realization of a label free, real time, high-throughput and low cost biosensing platforms to detect molecular interactions with a high level of sensitivity has been yet stunted due to two factors: one, slow binding kinetics caused by the lack of probe molecules on the sensors and two, limited mass transport due to the planar structure (two-dimensional) of the current biosensors. Here we present a novel three-dimensional (3D), highly sensitive, real-time, inexpensive and label-free nanotip array as a rapid and direct platform to sequence-specific DNA screening. Our nanotip sensors are designed to have a nano sized thin film as their sensing area (~ 20 nm), sandwiched between two sensing electrodes. The tip is then conjugated to a DNA oligonucleotide complementary to the sequence of interest, which is electrochemically detected in real-time via impedance changes upon the formation of a double-stranded helix at the sensor interface. This 3D configuration is specifically designed to improve the biomolecular hit rate and the detection speed. We demonstrate that our nanotip array effectively detects oligonucleotides in a sequence-specific and highly sensitive manner, yielding concentration-dependent impedance change measurements with a target concentration as low as 10 pM and discrimination against even a single mismatch. Notably, our nanotip sensors achieve this accurate, sensitive detection without relying on signal indicators or enhancing molecules like fluorophores. It can also easily be scaled for highly multiplxed detection with up to 5000 sensors/square centimeter, and integrated into microfluidic devices. The versatile, rapid, and sensitive performance of the nanotip array makes it an excellent candidate for point-of-care diagnostics, and high-throughput DNA analysis applications.

Nanoelectronic impedance detection of target cells.

Detection of cells is typically performed using optical fluorescence based techniques such as flow cytometry. Here we present the impedance detection of target cells using a nanoelectronic probe we have developed, which we refer to as the nanoneedle biosensor. The nanoneedle consists of a thin film conducting electrode layer at the bottom, an insulative oxide layer above, another conductive electrode layer above, and a protective oxide above. The electrical impedance is measured between the two electrode layers. Cells captured on the surface of the nanoneedle tip results in a decrease in the impedance across the sensing electrodes. The basic mechanisms behind the electrical response of cells in solution under an applied alternating electrical field stems from modulation of the relative permittivity at the interface. In this paper we discuss, the circuit model, the nanofabrication, and the testing and characterization of the sensor. We demonstrate proof of concept for detection of yeast cells with specificity. We envision the sensor presented in this paper to be combined with microfluidic pre-concentration technologies to develop low cost point-of-care diagnostic assays for the clinical setting.

Simulation and fabrication of a new novel 3D injectable biosensor for high throughput genomics and proteomics in a lab-on-a-chip device.

Biosensors are used for the detection of biochemical molecules such as proteins and nucleic acids. Traditional techniques, such as enzyme-linked immuno-sorbent assay (ELISA), are sensitive but require several hours to yield a result and usually require the attachment of a fluorophore molecule to the target molecule. Micromachined biosensors that employ electrical detection are now being developed. Here we describe one such device, which is ultrasensitive, real-time, label free and localized. It is called the nanoneedle biosensor and shows promise to overcome some of the current limitations of biosensors. The key element of this device is a 10 nm wide annular gap at the end of the needle, which is the sensitive part of the sensor. The total diameter of the sensor is about 100 nm. Any change in the population of molecules in this gap results in a change of impedance across the gap. Single molecule detection should be possible because the sensory part of the sensor is in the range of bio-molecules of interest. To increase throughput we can flow the solution containing the target molecules over an array of such structures, each with its own integrated read-out circuitry to allow 'real-time' detection (i.e. several minutes) of label free molecules without sacrificing sensitivity. To fabricate the arrays we used electron beam lithography together with associated pattern transfer techniques. Preliminary measurements on individual needle structures in water are consistent with the design. Since the proposed sensor has a rigid nano-structure, this technology, once fully developed, could ultimately be used to directly monitor protein quantities within a single living cell, an application that would have significant utility for drug screening and studying various intracellular signaling pathways.

Erratum: Publisher's Note: "Label-free electronic probing of nucleic acids and proteins at the nanoscale using the nanoneedle biosensor" [Biomicrofluidics 7, 044114 (2013)].

[This corrects the article on p. 044114 in vol. 7.].

Microneedle Biosensor: A Method for Direct Label-free Real Time Protein Detection.

Here we present the development of an array of electrical micro-biosensors in a microfluidic channel, called microneedle biosensors. A microneedle biosensor is a real-time, label-free, direct electrical detection platform, which is capable of high sensitivity detection, measuring the change in ionic current and impedance modulation, due to the presence or reaction of biomolecules such as proteins and nucleic acids. In this study, we successfully fabricated and electrically characterized the sensors and demonstrated successful detection of target protein. In this study, we used biotinylated bovine serum albumin as the receptor and streptavidin as the target analyte.

Label-free electronic probing of nucleic acids and proteins at the nanoscale using the nanoneedle biosensor.

Detection of proteins and nucleic acids is dominantly performed using optical fluorescence based techniques, which are more costly and timely than electrical detection due to the need for expensive and bulky optical equipment and the process of fluorescent tagging. In this paper, we discuss our study of the electrical properties of nucleic acids and proteins at the nanoscale using a nanoelectronic probe we have developed, which we refer to as the Nanoneedle biosensor. The nanoneedle consists of four thin film layers: a conductive layer at the bottom acting as an electrode, an oxide layer on top, and another conductive layer on top of that, with a protective oxide above. The presence of proteins and nucleic acids near the tip results in a decrease in impedance across the sensing electrodes. There are three basic mechanisms behind the electrical response of DNA and protein molecules in solution under an applied alternating electrical field. The first change stems from modulation of the relative permittivity at the interface. The second mechanism is the formation and relaxation of the induced dipole moment. The third mechanism is the tunneling of electrons through the biomolecules. The results presented in this paper can be extended to develop low cost point-of-care diagnostic assays for the clinical setting.