Investigating nano-sized tumor-derived extracellular vesicles in enhancing anti-PD-1 immunotherapy.

Anti-PD1 immune checkpoint blockade (ICB) has shown promising results for treating several aggressive cancers, enhancing patient survival rates. The variability in clinical response to anti-PD1 ICB is thought to be driven by patient-specific biology and heterogeneity within the tumor microenvironment. Tumor-derived extracellular vesicles (TDEVs), nano-sized particles released from tumor cells, can modulate the tumor microenvironment, leading to immunosuppression and tumor progression. Hence, TDEVs may contribute to the variability in treatment response and play a crucial role in the failure of anti-PD1 immunotherapy. In this study, we develop a systems biology approach to interrogate the role of TDEVs on the response dynamics for anti-PD1 blockade. Our results suggest that the detection and profiling of TDEVs can help screen patients for anti-PD-1 immunotherapy. Moreover, the results in this study suggest that TDEVs and IL-12 can potentially be liquid biopsy biomarkers to profile patient response to anti-PD1 ICB and tailor patient-specific treatment protocols. Importantly, the methodology is generalizable to other types of cancer immunotherapies. Therefore, the collection of patient-specific liquid biopsy data, and the implementation of those data into the systems biology framework, may offer the opportunity to discover new biomarkers for patient drug screening and enable the continuous monitoring of patient response to treatment and adaptation of patient-specific immunotherapy treatment protocols to overcome therapeutic resistance.

Rapid miRNA detection in skin interstitial fluid using a hydrogel microneedle patch integrated with DNA probes and graphene oxide.

MicroRNA (miRNA) is a type of short, non-coding nucleic acid molecule that plays essential roles in diagnosing and prognosing various types of cancer. MiRNA is abundantly present in skin interstitial fluid (ISF), providing real-time and localized physiological information. Hydrogel microneedle (HMN) patches enable miRNA collection in a fast, pain-free, minimally invasive, and user-friendly manner. In this study, we introduced a fluorescence-based HMN assay, namely the HMN-miR sensor, composed of methacrylated hyaluronic acid (MeHA) and a graphene oxide-probe DNA (GO.pDNA) conjugate for miR21 and miR210 detection. The HMN-miR sensor demonstrates excellent skin penetration efficiency, rapid ISF collection capability, and sufficient miRNA detection and sequence identification specificity. The HMN-miR sensor facilitates a new assay that, with further optimization, could be applied in future clinical settings. Its simple fabrication process and excellent biocompatibility give it significant potential for various clinical uses, such as personalized cancer treatment and monitoring the healing progress of burn wounds.

Integrated Electrochemical Aptamer Biosensing and Colorimetric pH Monitoring via Hydrogel Microneedle Assays for Assessing Antibiotic Treatment.

Current methods for therapeutic drug monitoring (TDM) have a long turnaround time as they involve collecting patients' blood samples followed by transferring the samples to medical laboratories where sample processing and analysis are performed. To enable real-time and minimally invasive TDM, a microneedle (MN) biosensor to monitor the levels of two important antibiotics, vancomycin (VAN) and gentamicin (GEN) is developed. The MN biosensor is composed of a hydrogel MN (HMN), and an aptamer-functionalized flexible (Flex) electrode, named HMN-Flex. The HMN extracts dermal interstitial fluid (ISF) and transfers it to the Flex electrode where sensing of the target antibiotics happens. The HMN-Flex performance is validated ex vivo using skin models as well as in vivo in live rat animal models. Data is leveraged from the HMN-Flex system to construct pharmacokinetic profiles for VAN and GEN and compare these profiles with conventional blood-based measurements. Additionally, to track pH and monitor patient's response during antibiotic treatment, an HMN is developed that employs a colorimetric method to detect changes in the pH, named HMN-pH assay, whose performance has been validated both in vitro and in vivo. Further, multiplexed antibiotic and pH detection is achieved by simultaneously employing the HMN-pH and HMN-Flex on live animals.

Multifunctional Dopamine-Based Hydrogel Microneedle Electrode for Continuous Ketone Sensing.

Diabetic ketoacidosis (DKA), a severe complication of type 1 diabetes (T1D), is triggered by production of large quantities of ketone bodies, requiring patients with T1D to constantly monitor their ketone levels. Here, a skin-compatible hydrogel microneedle (HMN)-continuous ketone monitoring (HMN-CKM) device is reported. The sensing mechanism relies on the catechol-quinone chemistry inherent to the dopamine (DA) molecules that are covalently linked to the polymer structure of the HMN patch. The DA serves the dual-purpose of acting as a redox mediator for measuring the byproduct of oxidation of 3-beta-hydroxybutyrate (ß-HB), the primary ketone bodies; while, also facilitating the formation of a crosslinked HMN patch. A universal approach involving pre-oxidation and detection of the generated catechol compounds is introduced to correlate the sensor response to the ß-HB concentrations. It is further shown that real-time tracking of a decrease in ketone levels of T1D rat model is possible using the HMN-CKM device, in conjunction with a data-driven machine learning model that considers potential time delays.

Wearable Aptalyzer Integrates Microneedle and Electrochemical Sensing for In Vivo Monitoring of Glucose and Lactate in Live Animals.

Continuous monitoring of clinically relevant biomarkers within the interstitial fluid (ISF) using microneedle (MN)-based assays, has the potential to transform healthcare. This study introduces the Wearable Aptalyzer, an integrated system fabricated by combining biocompatible hydrogel MN arrays for ISF extraction with an electrochemical aptamer-based biosensor for in situ monitoring of blood analytes. The use of aptamers enables continuous monitoring of a wide range of analytes, beyond what is possible with enzymatic monitoring. The Wearable Aptalyzer is used for real-time and multiplexed monitoring of glucose and lactate in ISF. Validation experiments using live mice and rat models of type 1 diabetes demonstrate strong correlation between the measurements collected from the Wearable Aptalyzer in ISF and those obtained from gold-standard techniques for blood glucose and lactate, for each analyte alone and in combination. The Wearable Aptalyzer effectively addresses the limitations inherent in enzymatic detection methods as well as solid MN biosensors and the need for reliable and multiplexed bioanalytical monitoring in vivo.

Microneedle Assays for Continuous Health Monitoring: Challenges and Solutions.

Continuous health monitoring aims to reduce hospitalization and the need for constant supervision of the patients. For an outpatient monitoring device to be effective, it must meet certain criteria: it should demand minimal patient involvement, be reliable, be connected, remain stable with infrequent replacements, be cost-efficient, be compatible with humans, and ultimately be self-powered. Microneedle (MN) technology, designed for transdermal biosensing, offers a promising solution for meeting a wide range of these demands in the field of continuous health monitoring. A variety of MN platforms have been developed to facilitate this crucial function. Our focus in this Perspective is on the significant challenges linked to MN-based biosensors. These challenges include ensuring skin compatibility, the effective integration of biorecognition elements into the MN systems, and the durability concerns of these sensors in enabling extended periods of continuous monitoring. Tackling these hurdles could pave the way for more effective and reliable MN-based health monitoring solutions in the future.

Experimental measurement and numerical modeling of deformation behavior of breast cancer cells passing through constricted microfluidic channels.

During the multistep process of metastasis, cancer cells encounter various mechanical forces which make them deform drastically. Developing accurate in-silico models, capable of simulating the interactions between the mechanical forces and highly deformable cancer cells, can pave the way for the development of novel diagnostic and predictive methods for metastatic progression. Spring-network models of cancer cell, empowered by our recently proposed identification approach, promises a versatile numerical tool for developing experimentally validated models that can simulate complex interactions at cellular scale. Using this numerical tool, we presented spring-network models of breast cancer cells that can accurately replicate the experimental data of deformation behavior of the cells flowing in a fluidic domain and passing narrow constrictions comparable to microcapillary. First, using high-speed imaging, we experimentally studied the deformability of breast cancer cell lines with varying metastatic potential (MCF-7 (less invasive), SKBR-3 (medium-high invasive), and MDA-MB-231 (highly invasive)) in terms of their entry time to a constricted microfluidic channel. We observed that MDA-MB-231, that has the highest metastatic potential, is the most deformable cell among the three. Then, by focusing on this cell line, experimental measurements were expanded to two more constricted microchannel dimensions. The experimental deformability data in three constricted microchannel sizes for various cell sizes, enabled accurate identification of the unknown parameters of the spring-network model of the breast cancer cell line (MDA-MB-231). Our results show that the identified parameters depend on the cell size, suggesting the need for a systematic procedure for identifying the size-dependent parameters of spring-network models of cells. As the numerical results show, the presented cell models can simulate the entry process of the cell into constricted channels with very good agreements with the measured experimental data.

A competitive, bead-based assay combined with microfluidics for multiplexed toxin detection.

The requirement for rapid, in-field detection of cyanotoxins in water resources necessitates the developing of an easy-to-use and miniaturized system for their detection. We present a novel bead-based, competitive fluorescence assay for multiplexed detection of two types of toxins: microcystin-LR (MC-LR) and okadaic acid (OA). To automate the detection process, a reusable microfluidic device, termed toxin-chip, was designed and validated. The toxin-chip consists of a micromixer where the target toxins were efficiently mixed with a reagent solution, and a detection chamber for magnetic retainment of beads for downstream analysis. Quantum dots (QDs) were used as the reporter molecules to enhance the sensitivity of the assay and the emitted fluorescence signal from QDs was reversely proportional to the amount of toxins in the solution. An image analysis program was also developed to further automate the detection and analysis steps. Two toxins were simultaneously analyzed on a single microfluidic chip, and the device exhibited a low detection limit of 10-4 µg ml-1 for MC-LR and 4 × 10-5 µg ml-1 for OA detection. The bead-based, competitive assay also showed remarkable chemical specificity against potential interfering toxins. We also validated the device performance using natural lake water samples from Sunfish Lake of Waterloo. The toxin-chip holds promise as a versatile and simple quantification tool for cyanotoxin detection, with the potential of detecting more toxins.

A Hydrogel Microneedle Assay Combined with Nucleic Acid Probes for On-Site Detection of Small Molecules and Proteins.

Point-of-care testing (POCT) of clinical biomarkers is critical to health monitoring and timely treatment, yet biosensing assays capable of detecting biomarkers without the need for costly external equipment and reagents are limited. Blood-based assays are, specifically, challenging as blood collection is invasive and follow-upprocessing is required. Here, we report a versatile assay that employs hydrogel microneedles (HMNs) to extract interstitial fluid (ISF), in a minimally invasive manner integrated with graphene oxide-nucleic acid (GO.NA)-based fluorescence biosensor to sense the biomarkers of interest in situ. The HMN-GO.NA assay is supplemented with a portable detector, enabling a complete POCT procedure. Our system could successfully measure four clinically important biomarkers (glucose, uric acid (UA), insulin, and serotonin) ex vivo, in addition, to accurately detecting glucose and UA in vivo.

A microfluidic plasma separation device combined with a surface plasmon resonance biosensor for biomarker detection in whole blood.

Biomarker detection in whole blood enables understanding of the cause, progression, relapse or outcome of treatment of a disease. Conventional biomarker detection techniques, such as enzyme-linked immunosorbent assay, polymerase chain reaction, and immunofluorescence, require long assay time, costly laboratory instruments, large reagent volume and sample pre-processing. Hence, there is an unmet need for reliable capture and detection of biomarkers in unprocessed blood which are adaptable to point-of-care (POC) testing. Here, we present a simple, low-cost, and rapid protein detection device from whole blood samples which has the potential to be employed in a POC setting. The platform consists of two components: a plasma separation device that extracts plasma from whole blood without the application of any external active forces and a SPR sensor chip that uses a label-free optical technique for the detection of biomarkers in the extracted plasma. We have demonstrated the detection of IgG and IgM biomolecules in unprocessed blood at concentrations lower than the physiological value within 15 min. The proposed technique has the potential for improving the diagnosis and screening of many diseases, including cancer, influenza, human immunodeficiency virus, and SARS-Cov2 at POC.

A Conductive Hydrogel Microneedle-Based Assay Integrating PEDOT:PSS and Ag-Pt Nanoparticles for Real-Time, Enzyme-Less, and Electrochemical Sensing of Glucose.

Continuous glucose meters (CGMs) have tremendously boosted diabetes care by emancipating millions of diabetic patients' need for repeated self-testing by pricking their fingers every few hours. However, CGMs still suffer from major deficiencies regarding accuracy, precision, and stability. This is mainly due to their dependency on an enzymatic detection mechanism. Here a low-cost hydrogel microneedle (HMN)-CGM assay fabricated using swellable dopamine (DA)-hyaluronic acid (HA) hydrogel for glucose interrogation in dermal interstitial fluid (ISF) is introduced. Platinum and silver nanoparticles are synthesized within the 3D porous hydrogel scaffolds for nonenzymatic electrochemical sensing of the glucose. Incorporation of a highly water dispersible conductive polymer, poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) enhances the electrical properties of HMN array, making the patch suitable as the working electrode of the sensor. The in vitro and ex vivo characterization of this newly developed HMN patch is fully studied. The performance of the HMN-CGM for real-time measurement of glucose is also shown using a rat model of type 1 diabetes. The device introduces the first HMN-based assay for tracking important disease biomarkers and expect to pave the way for next generation of polymeric-based sensors.

Sub-terahertz silicon-based on-chip absorption spectroscopy using thin-film model for biological applications.

Spectroscopy in the sub-terahertz (sub-THz) range of frequencies has been utilized to study the picosecond dynamics and interaction of biomolecules. However, widely used free-space THz spectrometers are typically limited in their functionality due to low signal-to-noise ratio and complex setup. On-chip spectrometers can revolutionize THz spectroscopy allowing integration, compactness, and low-cost fabrication. In this paper, a low-loss silicon-based platform is proposed for on-chip sub-THz spectroscopy. Through functionalization of silicon chip and immobilization of bio-particles, we demonstrate the ability to characterize low-loss nano-scale biomolecules across the G-band (0.14-0.22 THz). We also introduce an electromagnetic thin-film model to account for the loading effect of the immobilized biomolecules, i.e. dehydrated streptavidin and immunoglobulin antibody, as two key molecules in the biosensing discipline. The proposed platform was fabricated using a single mask micro-fabrication process, and then measured by a vector network analyzer (VNA), which offers high dynamic range and high spectral resolution measurements. The proposed planar platform is general and paves the way towards low-loss, cost-effective and integrated sub-THz biosensors for the detection and characterization of biomolecules.

A Conductive Hydrogel-Based Microneedle Platform for Real-Time pH Measurement in Live Animals.

Conventional microneedles (MNs) have been extensively reported and applied toward a variety of biosensing and drug delivery applications. Hydrogel forming MNs with the added ability to electrically track health conditions in real-time is an area yet to be explored. The first conductive hydrogel microneedle (HMN) electrode that is capable of on-needle pH detection with no postprocessing required is presented here. The HMN array is fabricated using a swellable dopamine (DA) conjugated hyaluronic acid (HA) hydrogel, and is embedded with poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PEDOT:PSS) to increase conductivity. The catechol-quinone chemistry intrinsic to DA is used to measure pH in interstitial fluid (ISF). The effect of PEDOT:PSS on the characteristics of the HMN array such as swelling capability and mechanical strength is fully studied. The HMN's capability for pH measurement is first demonstrated using porcine skin equilibrated with different pH solutions ranging from 3.5 to 9. Furthermore, the HMN-pH meter is capable of in vivo measurements with a 93% accuracy compared to a conventional pH probe meter. This HMN technology bridges the gap between traditional metallic electrochemical biosensors and the direct extraction of ISF, and introduces a platform for the development of polymeric wearable sensors capable of on-needle detection.

Hydrogel Microneedle-Assisted Assay Integrating Aptamer Probes and Fluorescence Detection for Reagentless Biomarker Quantification.

Analyzing interstitial fluid (ISF) via microneedle (MN) devices enables patient health monitoring in a minimally invasive manner and in point-of-care settings. However, most MN-based diagnostic approaches require complicated fabrication processes and postprocessing of the extracted ISF or are limited to detection of electrochemically active biomarkers. Here, we show on-needle measurement of target analytes by integrating hydrogel microneedles with aptamer probes as the recognition elements. Fluorescently tagged aptamer probes are chemically attached to the hydrogel matrix using a simple and novel approach, while a cross-linked patch is formed. For reagentless detection, we employ a strand displacement strategy where fluorophore-conjugated aptamers are hybridized with a DNA competitor strand conjugated to a quencher molecule. The assay is utilized for rapid (2 min) measurement of glucose, adenosine triphosphate, l-tyrosinamide, and thrombin ex vivo. Furthermore, the system enables specific and sensitive quantification of rising and falling concentrations of glucose in an animal model of diabetes to track hypoglycemia, euglycemia, and hyperglycemia conditions. Our assay can be applied for rapid measurement of a diverse range of biomarkers, proteins, or small molecules, introducing a generalizable platform for biomolecule quantification, and has the potential to improve the quality of life of patients who are in need of close monitoring of biomarkers of health and disease.

An integrated microfluidic electrochemical assay for cervical cancer detection at point-of-care testing.

Cervical cancer (CC) is a major health care problem in low- and middle-income countries, necessitating the development of low-cost and easy-to-use assays for CC detection at point-of-care (POC) settings. An integrated microfluidic electrochemical assay for CC detection, named IMEAC, is presented that has the potential for identifying CC circulating DNA in whole blood samples. The IMEAC consists of two main modules: a plasma separator device that isolates plasma from whole blood with high purity and without the need for any external forces connected to a graphene oxide-based electrochemical biosensor that uses specific probe molecules for the detection of CC circulating DNA molecules. We fully characterize the performance of the individual modules and show that the integrated assay can be utilized for target DNA detection in whole blood samples, thus potentially transforming CC detection and screening at remote locations.

Recent Advances in Device Engineering and Computational Analysis for Characterization of Cell-Released Cancer Biomarkers.

During cancer progression, tumors shed different biomarkers into the bloodstream, including circulating tumor cells (CTCs), extracellular vesicles (EVs), circulating cell-free DNA (cfDNA), and circulating tumor DNA (ctDNA). The analysis of these biomarkers in the blood, known as 'liquid biopsy' (LB), is a promising approach for early cancer detection and treatment monitoring, and more recently, as a means for cancer therapy. Previous reviews have discussed the role of CTCs and ctDNA in cancer progression; however, ctDNA and EVs are rapidly evolving with technological advancements and computational analysis and are the subject of enormous recent studies in cancer biomarkers. In this review, first, we introduce these cell-released cancer biomarkers and briefly discuss their clinical significance in cancer diagnosis and treatment monitoring. Second, we present conventional and novel approaches for the isolation, profiling, and characterization of these markers. We then investigate the mathematical and in silico models that are developed to investigate the function of ctDNA and EVs in cancer progression. We convey our views on what is needed to pave the way to translate the emerging technologies and models into the clinic and make the case that optimized next-generation techniques and models are needed to precisely evaluate the clinical relevance of these LB markers.

Computational and Experimental Model to Study Immunobead-Based Assays in Microfluidic Mixing Platforms.

In immunobead-based assays, micro/nanobeads are functionalized with antibodies to capture the target analytes, which can significantly improve the assay's performance. The immunobead-based assays have been recently combined with microfluidic mixing devices and customized for a variety of applications. However, device design and process optimization to achieve the best performance remain a substantial technological challenge. Here, we introduce a computational model that enables the rational design and optimization of the immunobead-based assay in a microfluidic mixing channel. We use numerical methods to examine the effect of the flow rates, channel geometry, bead's trajectory, and the analyte and reagent characteristics on the efficiency of analyte capture on the surface of microbeads. This model accounts for different bead movements inside the microchannel, with the goal of simulating an actual active binding environment. The model is further validated experimentally where different microfluidic channels are tested to capture the target analytes. Our experimental results are shown to meet theoretical predictions. While the model is demonstrated here for the analysis of IgG capture in simple and herringbone-structured microchannels, it can be readily adapted to a broad range of target molecules and different device designs.

A microfluidic platform enables comprehensive gene expression profiling of mouse retinal stem cells.

Loss of photoreceptors due to retinal degeneration is a major cause of untreatable visual impairment and blindness. Cell replacement therapy, using retinal stem cell (RSC)-derived photoreceptors, holds promise for reconstituting damaged cell populations in the retina. One major obstacle preventing translation to the clinic is the lack of validated markers or strategies to prospectively identify these rare cells in the retina and subsequently enrich them. Here, we introduce a microfluidic platform that combines nickel micromagnets, herringbone structures, and a design enabling varying flow velocities among three compartments to facilitate a highly efficient enrichment of RSCs. In addition, we developed an affinity enrichment strategy based on cell-surface markers that was utilized to isolate RSCs from the adult ciliary epithelium. We showed that targeting a panel of three cell surface markers simultaneously facilitates the enrichment of RSCs to 1 : 3 relative to unsorted cells. Combining the microfluidic platform with single-cell whole-transcriptome profiling, we successfully identified four differentially expressed cell surface markers that can be targeted simultaneously to yield an unprecedented 1 : 2 enrichment of RSCs relative to unsorted cells. We also identified transcription factors (TFs) that play functional roles in maintenance, quiescence, and proliferation of RSCs. This level of analysis for the first time identified a spectrum of molecular and functional properties of RSCs.

A fluorescence sandwich immunoassay for the real-time continuous detection of glucose and insulin in live animals.

Biosensors that continuously measure circulating biomolecules in real time could provide insights into the health status of patients and their response to therapeutics. But biosensors for the continuous real-time monitoring of analytes in vivo have only reached nanomolar sensitivity and can measure only a handful of molecules, such as glucose and blood oxygen. Here we show that multiple analytes can be continuously and simultaneously measured with picomolar sensitivity and sub-second resolution via the integration of aptamers and antibodies into a bead-based fluorescence sandwich immunoassay implemented in a custom microfluidic chip. After an incubation time of 30 s, bead fluorescence is measured using a high-speed camera under spatially multiplexed two-colour laser illumination. We used the assay for continuous quantification of glucose and insulin concentrations in the blood of live diabetic rats to resolve inter-animal differences in the pharmacokinetic response to insulin as well as discriminate pharmacokinetic profiles from different insulin formulations. The assay can be readily modified to continuously and simultaneously measure other blood analytes in vivo.

Peptide-Functionalized Nanostructured Microarchitectures Enable Rapid Mechanotransductive Differentiation.

Microenvironmental factors play critical roles in regulating stem cell fate, providing a rationale to engineer biomimetic microenvironments that facilitate rapid and effective stem cell differentiation. Three-dimensional (3D) hierarchical microarchitectures have been developed to enable rapid neural differentiation of multipotent human mesenchymal stromal cells (HMSCs) via mechanotransduction. However, low cell viability during long-term culture and poor cell recovery efficiency from the architectures were also observed. Such problems hinder further applications of the architectures in stem cell differentiation. Here, we present improved 3D nanostructured microarchitectures functionalized with cell-adhesion-promoting arginylglycylaspartic acid (RGD) peptides. These RGD-functionalized architectures significantly upregulated long-term cell viability and facilitated effective recovery of differentiated cells from the architectures while maintaining high differentiation efficiency. Efficient recovery of highly viable differentiated cells enabled the downstream analysis of morphology and protein expression to be performed. Remarkably, even after the removal of the mechanical stimulus provided by the 3D microarchitectures, the recovered HMSCs showed a neuron-like elongated morphology for 10 days and consistently expressed microtubule-associated protein 2, a mature neural marker. RGD-functionalized nanostructured microarchitectures hold great potential to guide effective differentiation of highly viable stem cells.