On-chip hybrid integration of swept frequency distributed-feedback laser with silicon photonic circuits using photonic wire bonding.

This paper presents a novel co-packaging approach through on-chip hybrid laser integration with photonic circuits using photonic wire bonding. The process involves die-bonding a low-cost semiconductor distributed-feedback (DFB) laser into a deep trench on a silicon-on-insulator (SOI) chip and coupling it to the silicon circuitry through photonic wire bonding (PWB). After characterizing the power-current-voltage (LIV) and optical spectrum of the laser, a wavelength-current relationship utilizing its tunability through self-heating a swept-frequency laser (SFL) is developed. Photonic integrated circuit (PIC) resonators are successfully characterized using the SFL method, demonstrating signal detection with a quality factor comparable to measurements conducted with an off-chip benchtop laser.

An improved in vitro model for studying the structural and functional properties of the endothelial glycocalyx in arteries, capillaries and veins.

The endothelial glycocalyx is a dynamic structure integral to blood vessel hemodynamics and capable of tightly regulating a range of biological processes (ie, innate immunity, inflammation, and coagulation) through dynamic changes in its composition of the brush structure. Evaluating the specific roles of the endothelial glycocalyx under a range of pathophysiologic conditions has been a challenge in vitro as it is difficult to generate functional glycocalyces using commonly employed 2D cell culture models. We present a new multi-height microfluidic platform that promotes the growth of functional glycocalyces by eliciting unique shear stress forces over a continuous human umbilical vein endothelial cell monolayer at magnitudes that recapitulate the physical environment in arterial, capillary and venous regions of the vasculature. Following 72 hours of shear stress, unique glycocalyx structures formed within each region that were distinct from that observed in short (3 days) and long-term (21 days) static cell culture. The model demonstrated glycocalyx-specific properties that match the characteristics of the endothelium in arteries, capillaries and veins, with respect to surface protein expression, platelet adhesion, lymphocyte binding and nanoparticle uptake. With artery-to-capillary-to-vein transition on a continuous endothelial monolayer, this in vitro platform is an improved system over static cell culture for more effectively studying the role of the glycocalyx in endothelial biology and disease.

High-performance sub-wavelength grating-based resonator sensors with substrate overetch.

We propose a strategy to improve sensing performance of sub-wavelength-grating (SWG) waveguide-based sensors by introducing a substrate-overetch (SOE) geometry. The proposed SOE-SWG waveguide shows enhanced analyte interaction and a reduced group index, which improves the sensitivity of resonator-based sensors. The SiO2 overetch process was realized in Ar/C4F8/O2 plasma for 40 sec with a SiO2/Si selectivity of 10:1, obtaining a 285-nm anisotropic overetch in the SiO2 layer. Sensor performance of the SOE-SWG architecture is characterized by using isopropyl alcohol solutions, indicating an enhanced bulk sensitivity up to 575 nm/RIU (479 nm/RIU before the SOE), and a maximum waveguide mode sensitivity larger than one.

Erratum: Luan, E.X.; Shoman, H.; Ratner, D.M.; Cheung, K.C.; Chrostowski, L. Silicon Photonic Biosensors Using Label-Free Detection. Sensors 2018, 18, 3519.

The authors wish to make the following corrections in their published paper in Sensors [...].

A novel approach to producing uniform 3-D tumor spheroid constructs using ultrasound treatment.

Producing three-dimensional (3-D) multicellular tumor spheroids (TSs) is valuable for characterizing anticancer drugs since they provide a more representative model of the 3-D in vivo tumor than conventional two-dimensional (2-D) monolayer culture. The interaction of tumor cells with the extracellular matrix (ECM) in a 3-D culture environment is more similar to a tumor in vivo than in a 2-D environment; cell-cell and cell-ECM interaction can influence cell behaviour, such as in response to drug treatment. In vitro tumor spheroid models have been developed using microfluidic systems to generate 3-D hydrogel beads containing components of alginate and ECM protein, such as collagen, with high uniformity and throughput. Cell-laden hydrogel droplets are formed using a flow focusing process wherein the hydrogel precursors should be a homogeneous mixture. During gelation of the droplets into beads, the alginate acts as a fast gelling component helping to maintain the spherical shape of beads and preventing coalescence as the temperature-sensitive collagen I component gels more slowly. To produce uniform hydrogel droplets using the microfluidic flow focusing system, the mixtures must be homogeneous. However, collagen's sensitivity to temperature can lead to formation of chunks of collagen gel inside of the mixture, causing the mixture to become non-uniform and risking chip clogging. In order to overcome this limitation, previous approaches have used a cooling system during bead encapsulation while tumor cells were also present in the mixture, but this procedure can contribute to a delay in cell proliferation. Here a novel yet simple method is developed to prepare homogeneous pre-bead-encapsulation-mixtures containing collagen type I through ultrasonication. This method allows the cultivation of homogenous TS cultures with high uniformity and compact structure, and not only maintains cell viability but also the proliferation of cells in alginate/collagen hydrogel bead cultures. Depending on the sonication parameters, time and temperature, collagen can form small sized fibrils to thick fibers. Here, the mixtures containing collagen are assessed for morphology of collagen fibers/fibrils, cell viability, and proliferation. Human source Michigan Cancer Foundation-7 (MCF-7) breast cancer cells are successfully incorporated into alginate/collagen mixtures, followed by sonication, and then bead production. After bead gelation, the encapsulated MCF-7 cells remained viable and proliferated to form uniform TSs when the beads contained alginate and collagen. Results indicate that ultrasound treatment (UST) provides a powerful technique to change the structure of collagen from fiber to fibril, and to disperse collagen fibers in the mixture homogeneously for an application to generate uniform hydrogel beads and spheroids while not inhibiting cell proliferation.

Silicon Photonic Biosensors Using Label-Free Detection.

Thanks to advanced semiconductor microfabrication technology, chip-scale integration and miniaturization of lab-on-a-chip components, silicon-based optical biosensors have made significant progress for the purpose of point-of-care diagnosis. In this review, we provide an overview of the state-of-the-art in evanescent field biosensing technologies including interferometer, microcavity, photonic crystal, and Bragg grating waveguide-based sensors. Their sensing mechanisms and sensor performances, as well as real biomarkers for label-free detection, are exhibited and compared. We also review the development of chip-level integration for lab-on-a-chip photonic sensing platforms, which consist of the optical sensing device, flow delivery system, optical input and readout equipment. At last, some advanced system-level complementary metal-oxide semiconductor (CMOS) chip packaging examples are presented, indicating the commercialization potential for the low cost, high yield, portable biosensing platform leveraging CMOS processes.

Sub-wavelength grating for enhanced ring resonator biosensor.

While silicon photonic resonant cavities have been widely investigated for biosensing applications, enhancing their sensitivity and detection limit continues to be an area of active research. Here, we describe how to engineer the effective refractive index and mode profile of a silicon-on-insulator (SOI) waveguide using sub-wavelength gratings (SWG) and report on its observed performance as a biosensor. We designed a 30 µm diameter SWG ring resonator and fabricated it using Ebeam lithography. Its characterization resulted in a quality factor, Q, of 7 · 103, bulk sensitivity Sb = 490 nm/RIU, and system limit of detection sLoD = 2 · 10-6 RIU. Finally we employ a model biological sandwich assay to demonstrate its utility for biosensing applications.

Alginate core-shell beads for simplified three-dimensional tumor spheroid culture and drug screening.

We demonstrate that when using cell-laden core-shell hydrogel beads to support the generation of tumor spheroids, the shell structure reduces the out-of-bead and monolayer cell proliferation that occurs during long-term culture of tumor cells within core-only alginate beads. We fabricate core-shell beads in a two-step process using simple, one-layer microfluidic devices. Tumor cells encapsulated within the bead core will proliferate to form multicellular aggregates which can serve as three-dimensional (3-D) models of tumors in drug screening. Encapsulation in an alginate shell increased the time that cells could be maintained in three-dimensional culture for MCF-7 breast cancer cells prior to out-of-bead proliferation, permitting formation of spheroids over a period of 14 days without the need move the cell-laden beads to clean culture flasks to separate beads from underlying monolayers. Tamoxifen and docetaxel dose response shows decreased toxicity for multicellular aggregates in three-dimensional core-shell bead culture compared to monolayer. Using simple core-only beads gives mixed monolayer and 3-D culture during drug screening, and alters the treatment result compared to the 3-D core-shell or the 2-D monolayer groups, as measured by standard proliferation assay. By preventing the out-of-bead proliferation and subsequent monolayer formation that is observed with core-only beads, the core-shell structure can obviate the requirement to transfer the beads to a new culture flask during drug screening, an important consideration for cell-based drug screening and drugs which have high multicellular resistance index.

Designing a Microfluidic Device with Integrated Ratiometric Oxygen Sensors for the Long-Term Control and Monitoring of Chronic and Cyclic Hypoxia.

Control of oxygen over cell cultures in vitro is a topic of considerable interest, as chronic and cyclic hypoxia can alter cell behaviour. Both static and transient hypoxic levels have been found to affect tumour cell behaviour; it is potentially valuable to include these effects in early, in vitro stages of drug screening. A barrier to their inclusion is that rates of transient hypoxia can be a few cycles/hour, which is difficult to reproduce in traditional in vitro cell culture environments due to long diffusion distances from control gases to the cells. We use a gas-permeable three-layer microfluidic device to achieve spatial and temporal oxygen control with biologically-relevant switching times. We measure the oxygen profiles with integrated, ratiometric optical oxygen sensors, demonstrate sensor and system stability over multi-day experiments, and characterize a pre-bleaching process to improve sensor stability. We show, with both finite-element modelling and experimental data, excellent control over the oxygen levels by the device, independent of fluid flow rate and oxygenation for the operating flow regime. We measure equilibration times of approximately 10 min, generate complex, time-varying oxygen profiles, and study the effects of oxygenated media flow rates on the measured oxygen levels. This device could form a useful tool for future long-term studies of cell behaviour under hypoxia.

A simple method for eliminating fixed-region interference of aptamer binding during SELEX.

Standard libraries for systematic evolution of ligands by exponential enrichment (SELEX) typically utilize flanking regions that facilitate amplification of aptamers recovered from each selection round. Here, we show that these flanking sequences can bias the selection process, due in part to their ability to interfere with the fold or function of aptamers localized within the random region of the library sequence. We then address this problem by investigating the use of complementary oligonucleotides as a means to block aptamer interference by each flanking region. Isothermal titration calorimetry (ITC) studies are combined with fold predictions to both define the various interference mechanisms and assess the ability of added complementary oligonucleotides to ameliorate them. The proposed blocking strategy is thereby refined and then applied to standard library forms of benchmark aptamers against human α-thrombin, streptavidin, and vascular endothelial growth factor (VEGF). In each case, ITC data show that the new method effectively removes fixed-region mediated interference effects so that the natural binding affinity of the benchmark aptamer is completely restored. We further show that the binding affinities of properly functioning aptamers within a selection library are not affected by the blocking protocol, and that the method can be applied to various common library formats comprised of different flanking region sequences. Finally, we present a rapid and inexpensive qPCR-based method for determining the mean binding affinity of retained aptamer pools and use it to show that introduction of the pre-blocking method into the standard SELEX protocol results in retention of high-affinity aptamers that would otherwise be lost during the first round of selection. Significant enrichment of the available pool of high-affinity aptamers is thereby achieved in the first few rounds of selection. By eliminating single-strand (aptamer-like) structures within or involving the fixed regions, the technique is therefore shown to isolate aptamer sequence and function within the desired random region of the library members, and thereby provide a new selection method that is complementary to other available SELEX protocols.

Silicon photonic micro-disk resonators for label-free biosensing.

Silicon photonic biosensors are highly attractive for multiplexed Lab-on-Chip systems. Here, we characterize the sensing performance of 3 µm TE-mode and 10 µm dual TE/TM-mode silicon photonic micro-disk resonators and demonstrate their ability to detect the specific capture of biomolecules. Our experimental results show sensitivities of 26 nm/RIU and 142 nm/RIU, and quality factors of 3.3x10(4) and 1.6x10(4) for the TE and TM modes, respectively. Additionally, we show that the large disks contain both TE and TM modes with differing sensing characteristics. Finally, by serializing multiple disks on a single waveguide bus in a CMOS compatible process, we demonstrate a biosensor capable of multiplexed interrogation of biological samples.

Improving piezoelectric cell printing accuracy and reliability through neutral buoyancy of suspensions.

The sedimentation and aggregation of cells within inkjet printing systems has been hypothesized to negatively impact printer performance. The purpose of this study was to investigate this influence through the use of neutral buoyancy. Ficoll PM400 was used to create neutrally buoyant MCF-7 breast cancer cell suspensions, which were ejected using a piezoelectric drop-on-demand inkjet printing system. It was found that using a neutrally buoyant suspension greatly increased the reproducibility of consistent cell counts, and eliminated nozzle clogging. Moreover, the use of Ficoll PM400 was shown to not affect cellular viability. This is the first demonstration of such scale and accuracy achieved using a piezoelectric inkjet printing system for cellular dispensing.

Oxygen Measurement in Microdevices.

Oxygen plays a fundamental role in respiration and metabolism, and quantifying oxygen levels is essential in many environmental, industrial, and research settings. Microdevices facilitate the study of dynamic, oxygen-dependent effects in real time. This review is organized around the key needs for oxygen measurement in microdevices, including integrability into microfabricated systems; sensor dynamic range and sensitivity; spatially resolved measurements to map oxygen over two- or three-dimensional regions of interest; and compatibility with multimodal and multianalyte measurements. After a brief overview of biological readouts of oxygen, followed by oxygen sensor types that have been implemented in microscale devices and sensing mechanisms, this review presents select recent applications in organs-on-chip in vitro models and new sensor capabilities enabling oxygen microscopy, bioprocess manufacturing, and pharmaceutical industries. With the advancement of multiplexed, interconnected sensors and instruments and integration with industry workflows, intelligent microdevice-sensor systems including oxygen sensors will have further impact in environmental science, manufacturing, and medicine.

Biofunctionalization of Multiplexed Silicon Photonic Biosensors.

Silicon photonic (SiP) sensors offer a promising platform for robust and low-cost decentralized diagnostics due to their high scalability, low limit of detection, and ability to integrate multiple sensors for multiplexed analyte detection. Their CMOS-compatible fabrication enables chip-scale miniaturization, high scalability, and low-cost mass production. Sensitive, specific detection with silicon photonic sensors is afforded through biofunctionalization of the sensor surface; consequently, this functionalization chemistry is inextricably linked to sensor performance. In this review, we first highlight the biofunctionalization needs for SiP biosensors, including sensitivity, specificity, cost, shelf-stability, and replicability and establish a set of performance criteria. We then benchmark biofunctionalization strategies for SiP biosensors against these criteria, organizing the review around three key aspects: bioreceptor selection, immobilization strategies, and patterning techniques. First, we evaluate bioreceptors, including antibodies, aptamers, nucleic acid probes, molecularly imprinted polymers, peptides, glycans, and lectins. We then compare adsorption, bioaffinity, and covalent chemistries for immobilizing bioreceptors on SiP surfaces. Finally, we compare biopatterning techniques for spatially controlling and multiplexing the biofunctionalization of SiP sensors, including microcontact printing, pin- and pipette-based spotting, microfluidic patterning in channels, inkjet printing, and microfluidic probes.

PDMS Organ-On-Chip Design and Fabrication: Strategies for Improving Fluidic Integration and Chip Robustness of Rapidly Prototyped Microfluidic In Vitro Models.

The PDMS-based microfluidic organ-on-chip platform represents an exciting paradigm that has enjoyed a rapid rise in popularity and adoption. A particularly promising element of this platform is its amenability to rapid manufacturing strategies, which can enable quick adaptations through iterative prototyping. These strategies, however, come with challenges; fluid flow, for example, a core principle of organs-on-chip and the physiology they aim to model, necessitates robust, leak-free channels for potentially long (multi-week) culture durations. In this report, we describe microfluidic chip fabrication methods and strategies that are aimed at overcoming these difficulties; we employ a subset of these strategies to a blood-brain-barrier-on-chip, with others applied to a small-airway-on-chip. Design approaches are detailed with considerations presented for readers. Results pertaining to fabrication parameters we aimed to improve (e.g., the thickness uniformity of molded PDMS), as well as illustrative results pertaining to the establishment of cell cultures using these methods will also be presented.

An Optimization Framework for Silicon Photonic Evanescent-Field Biosensors Using Sub-Wavelength Gratings.

Silicon photonic (SiP) evanescent-field biosensors aim to combine the information-rich readouts offered by lab-scale diagnostics, at a significantly lower cost, and with the portability and rapid time to result offered by paper-based assays. While SiP biosensors fabricated with conventional strip waveguides can offer good sensitivity for label-free detection in some applications, there is still opportunity for improvement. Efforts have been made to design higher-sensitivity SiP sensors with alternative waveguide geometries, including sub-wavelength gratings (SWGs). However, SWG-based devices are fragile and prone to damage, limiting their suitability for scalable and portable sensing. Here, we investigate SiP microring resonator sensors designed with SWG waveguides that contain a "fishbone" and highlight the improved robustness offered by this design. We present a framework for optimizing fishbone-style SWG waveguide geometries based on numerical simulations, then experimentally measure the performance of ring resonator sensors fabricated with the optimized waveguides, targeting operation in the O-band and C-band. For the O-band and C-band devices, we report bulk sensitivities up to 349 nm/RIU and 438 nm/RIU, respectively, and intrinsic limits of detection as low as 5.1 × 10-4 RIU and 7.1 × 10-4 RIU, respectively. This performance is comparable to the state of the art in SWG-based sensors, positioning fishbone SWG resonators as an attractive, more robust, alternative to conventional SWG designs.

Airway-On-A-Chip: Designs and Applications for Lung Repair and Disease.

The lungs are affected by illnesses including asthma, chronic obstructive pulmonary disease, and infections such as influenza and SARS-CoV-2. Physiologically relevant models for respiratory conditions will be essential for new drug development. The composition and structure of the lung extracellular matrix (ECM) plays a major role in the function of the lung tissue and cells. Lung-on-chip models have been developed to address some of the limitations of current two-dimensional in vitro models. In this review, we describe various ECM substitutes utilized for modeling the respiratory system. We explore the application of lung-on-chip models to the study of cigarette smoke and electronic cigarette vapor. We discuss the challenges and opportunities related to model characterization with an emphasis on in situ characterization methods, both established and emerging. We discuss how further advancements in the field, through the incorporation of interstitial cells and ECM, have the potential to provide an effective tool for interrogating lung biology and disease, especially the mechanisms that involve the interstitial elements.

Review of Design Considerations for Brain-on-a-Chip Models.

In recent years, the need for sophisticated human in vitro models for integrative biology has motivated the development of organ-on-a-chip platforms. Organ-on-a-chip devices are engineered to mimic the mechanical, biochemical and physiological properties of human organs; however, there are many important considerations when selecting or designing an appropriate device for investigating a specific scientific question. Building microfluidic Brain-on-a-Chip (BoC) models from the ground-up will allow for research questions to be answered more thoroughly in the brain research field, but the design of these devices requires several choices to be made throughout the design development phase. These considerations include the cell types, extracellular matrix (ECM) material(s), and perfusion/flow considerations. Choices made early in the design cycle will dictate the limitations of the device and influence the end-point results such as the permeability of the endothelial cell monolayer, and the expression of cell type-specific markers. To better understand why the engineering aspects of a microfluidic BoC need to be influenced by the desired biological environment, recent progress in microfluidic BoC technology is compared. This review focuses on perfusable blood-brain barrier (BBB) and neurovascular unit (NVU) models with discussions about the chip architecture, the ECM used, and how they relate to the in vivo human brain. With increased knowledge on how to make informed choices when selecting or designing BoC models, the scientific community will benefit from shorter development phases and platforms curated for their application.

Droplet-based microfluidic system for multicellular tumor spheroid formation and anticancer drug testing.

Creating multicellular tumor spheroids is critical for characterizing anticancer treatments since it may provide a better model than monolayer culture of tumor cells. Moreover, continuous dynamic perfusion allows the establishment of long term cell culture and subsequent multicellular spheroid formation. A droplet-based microfluidic system was used to form alginate beads with entrapped breast tumor cells. After gelation, the alginate beads were trapped in microsieve structures for cell culture in a continuous perfusion system. The alginate environment permitted cell proliferation and the formation of multicellular spheroids was observed. The dose-dependent response of the tumor spheroids to doxorubicin, and anticancer drug, showed multicellular resistance compared to conventional monolayer culture. The microsieve structures maintain constant location of each bead in the same position throughout the device seeding process, cell proliferation and spheroid formation, treatment with drug, and imaging, permitting temporal and spatial tracking.

Microfluidic impedance-based flow cytometry.

Microfabricated flow cytometers can detect, count, and analyze cells or particles using microfluidics and electronics to give impedance-based characterization. Such systems are being developed to provide simple, low-cost, label-free, and portable solutions for cell analysis. Recent work using microfabricated systems has demonstrated the capability to analyze micro-organisms, erythrocytes, leukocytes, and animal and human cell lines. Multifrequency impedance measurements can give multiparametric, high-content data that can be used to distinguish cell types. New combinations of microfluidic sample handling design and microscale flow phenomena have been used to focus and position cells within the channel for improved sensitivity. Robust designs will enable focusing at high flowrates while reducing requirements for control over multiple sample and sheath flows. Although microfluidic impedance-based flow cytometers have not yet or may never reach the extremely high throughput of conventional flow cytometers, the advantages of portability, simplicity, and ability to analyze single cells in small populations are, nevertheless, where chip-based cytometry can make a large impact.