Ultra-low intensity light pulses for large cargo delivery into hard-to-transfect cells using an rGO mixed PDMS microtip device.

Nanoparticle-mediated photoporation has arisen as a universal intracellular delivery tool; however, the direct interaction of nanoparticles and cells hampers its clinical translation. Here, we report a uniform contactless intracellular delivery that transfects a large number of cells within a minute and avoids direct contact of nanoparticles and cells, thereby improving the cell viability. Our platform consists of an array of polydimethylsiloxane (PDMS) mixed reduced graphene oxide (rGO) nanoflakes on pyramidal microtips, uniformly distributed at the apex of the tip. The extraordinary optoelectronic properties of rGO were combined with micro-pyramidal cavities to entrap light in micro-cavities and efficiently convert it into heat through multiple reflections and absorptions. As a result, ultralow infra-red laser pulse irradiation could create cavitation bubbles followed by cell membrane deformation and biomolecular delivery. Using this delivery platform, we have achieved the delivery of small to large cargo (668 Da to 465 kDa) in various mammalian cells, including hard-to-transfect H9C2 cardiomyocytes. The best results were achieved for enzyme (465 kDa) delivery with a transfection efficiency and cell viability of 95% and 98%, respectively, in SiHa cells. The highly efficient cargo delivery tool demonstrated a safe and effective approach for cell therapy and diagnostics.

Direction-oriented fiber guiding with a tunable tri-layer-3D scaffold for periodontal regeneration.

Multilayered scaffolds mimicking mechanical and biological host tissue architectures are the current prerequisites for successful tissue regeneration. We propose our tunable tri-layered scaffold, designed to represent the native periodontium for potential regenerative applications. The fused deposition modeling platform is used to fabricate the novel movable three-layered polylactic acid scaffold mimicking in vivo cementum, periodontal ligament, and alveolar bone layers. The scaffold is further provided with multiple angulated fibers, offering directional guidance and facilitating the anchorage dependence on cell adhesion. Additionally, surface modifications of the scaffold were made by incorporating coatings like collagen and different concentrations of gelatin methacryloyl to enrich the cell adhesion and proliferation. The surface characterization of our designed scaffolds was performed using tribological studies, atomic force microscopy, contact angle measurement, scanning electron microscopy, and micro-computed tomography. Furthermore, the material characterization of this scaffold was investigated by attenuated total reflectance-Fourier transformed infrared spectroscopy. The scaffold's mechanical characterization, such as strength and compression modulus, was demonstrated by compression testing. The L929 mouse fibroblast cells and MG63 human osteosarcoma cells have been cultured on the scaffold. The scaffold's superior biocompatibility was evaluated using fluorescence dye with fluorescence microscopy, scanning electron microscopy, in vitro wound healing assay, MTT assay, and flow cytometry. The mineralization capability of the scaffolds was also studied. In conclusion, our study demonstrated the construction of a multilayered movable scaffold, which is highly biocompatible and most suitable for various downstream applications such as periodontium and in situ tissue regeneration of complex, multilayered tissues.

Bioglass and nano bioglass: A next-generation biomaterial for therapeutic and regenerative medicine applications.

Biomaterials are an indispensable component in tissue engineering that primarily functions to resemble the extracellular matrix of any tissue targeted for regeneration. In the last five decades, bioglass has been extensively used in the field of therapeutic and tissue engineering. The doping of metal components into bioglass and the synthesizing of nano bioglass particles have found remarkable implications, both in vivo and in vitro. These include various medical and biological applications such as rejuvenating tissues, facilitating regeneration, and delivering biomolecules into cells and therapy, etc. Therefore, the current review discusses the various techniques used in synthesizing bioglass particles, trends of various ion-doped nano bioglass, and their applications in therapy as well as in regenerative medicine, specifically in the fields of dentistry, cardiovascular, skin, nervous, and respiratory systems. Apart from these, this review also emphasizes the bioglass combined with diverse natural polymers (like collagen, chitosan, etc.) and their applications. Furthermore, we discuss the effectiveness of bioglass properties such as antibacterial effects, biomolecular delivery systems, tissue compatibility, and regenerative material. Finally, the prospects and limitations are elaborated.

Laser-Induced Intracellular Delivery: Exploiting Gold-Coated Spiky Polymeric Nanoparticles and Gold Nanorods under Near-Infrared Pulses for Single-Cell Nano-Photon-Poration.

This study explores the potential of laser-induced nano-photon-poration as a non-invasive technique for the intracellular delivery of micro/macromolecules at the single-cell level. This research proposes the utilization of gold-coated spiky polymeric nanoparticles (Au-PNPs) and gold nanorods (GNRs) to achieve efficient intracellular micro/macromolecule delivery at the single-cell level. By shifting the operating wavelength towards the near-infrared (NIR) range, the intracellular delivery efficiency and viability of Au-PNP-mediated photon-poration are compared to those using GNR-mediated intracellular delivery. Employing Au-PNPs as mediators in conjunction with nanosecond-pulsed lasers, a highly efficient intracellular delivery, while preserving high cell viability, is demonstrated. Laser pulses directed at Au-PNPs generate over a hundred hot spots per particle through plasmon resonance, facilitating the formation of photothermal vapor nanobubbles (PVNBs). These PVNBs create transient pores, enabling the gentle transfer of cargo from the extracellular to the intracellular milieu, without inducing deleterious effects in the cells. The optimization of wavelengths in the NIR region, coupled with low laser fluence (27 mJ/cm2) and nanoparticle concentrations (34 µg/mL), achieves outstanding delivery efficiencies (96%) and maintains high cell viability (up to 99%) across the various cell types, including cancer and neuronal cells. Importantly, sustained high cell viability (90-95%) is observed even 48 h post laser exposure. This innovative development holds considerable promise for diverse applications, encompassing drug delivery, gene therapy, and regenerative medicine. This study underscores the efficiency and versatility of the proposed technique, positioning it as a valuable tool for advancing intracellular delivery strategies in biomedical applications.

Microfluidic device-fabricated spiky nano-burflower shape gold nanomaterials facilitate large biomolecule delivery into cells using infrared light pulses.

Photothermal nanoparticle-sensitised photoporation is an emerging approach, which is considered an efficient tool for the intracellular delivery of biomolecules. Nevertheless, using this method to achieve high transfection efficiency generally compromises cell viability and uneven distribution of nanoparticles results in non-uniform delivery. Here, we show that high aspect ratio gold nano-burflowers, synthesised in a microfluidic device, facilitate highly efficient small to very-large cargo delivery uniformly using infrared light pulses without sacrificing cell viability. By precisely controlling the flow rates of shaping reagent and reducing agent, high-density (24 numbers) sharply branched spikes (∼80 nm tip-to-tip length) of higher aspect ratios (∼6.5) with a small core diameter (∼45 nm) were synthesised. As produced gold burflower-shape nanoparticles are biocompatible, colloidally stable (large surface zeta potential value), and uniform in morphology with a higher plasmonic peak (max. 890 nm). Theoretical analysis revealed that spikes on the nanoparticles generate a higher electromagnetic field enhancement upon interaction with light pulses. It induces plasmonic nanobubbles in the vicinity of the cells, followed by pore formation on the membrane leading to diverse biomolecular delivery into cells. Our platform has been successfully implemented for uniform delivery of small to very large biomolecules, including siRNA (20-24 bp), plasmid DNA expressing green fluorescent protein (6.2 kbp), Cas-9 plasmid (9.3 kbp), and ß-galactosidase enzyme (465 kDa) into diverse mammalian cells with high transfection efficiency and cell viability. For very large biomolecules such as enzymes, the best results were achieved as ∼100% transfection efficiency and ∼100% cell viability in SiHa cells. Together, our findings demonstrate that the spiky gold nano-burflower shape nanoparticles manufactured in a microfluidic system exhibited excellent plasmonic behaviour and could serve as an effective tool in manipulating cell physiology.

Ultrathin SU-8 membrane for highly efficient tunable cell patterning and massively parallel large biomolecular delivery.

Cell patterning is a powerful technique for the precise control and arrangement of cells, enabling detailed single-cell analysis with broad applications in therapeutics, diagnostics, and regenerative medicine. This study presents a novel and efficient technique that enables massively parallel high throughput cell patterning and precise delivery of small to large biomolecules into patterned cells. The innovative cell patterning device proposed in this study is a standalone, ultrathin 3D SU-8 micro-stencil membrane, with a thickness of 10 µm. It features an array of micro-holes ranging from 40 µm to 80 µm, spaced apart by 50 µm to 150 µm. By culturing cells on top of this SU-8 membrane, the technique achieves highly efficient cell patterns varying from single-cell to cell clusters on a Petri dish. Utilizing this technique, we have achieved a remarkable reproducible patterning efficiency for mouse fibroblast L929 (80.5%), human cervical SiHa (81%), and human neuroblastoma IMR32 (89.6%) with less than 1% defects in undesired areas. Single-cell patterning efficiency was observed to be highest at 75.8% for L929 cells. Additionally, we have demonstrated massively parallel high throughput uniform transfection of large biomolecules into live patterned cells by employing an array of titanium micro-rings (10 µm outer diameter, 3 µm inner diameter) activated through infrared light pulses. Successful delivery of a wide range of small to very large biomolecules, including propidium iodide (PI) dye (668.4 Da), dextran (3 kDa), siRNA (13.3 kDa), and ß-galactosidase enzyme (465 kDa), was accomplished in cell patterns for various cancer cells. Notably, our platform achieved exceptional delivery efficiencies of 97% for small molecules like PI dye and 84% for the enzyme, with corresponding high cell viability of 100% and 90%, respectively. Furthermore, the compact and reusable SU-8-based membrane device facilitates highly efficient cell patterning, transfection, and cell viability, making it a promising tool for diagnostics and therapeutic applications.

Massively Parallel High-Throughput Single-Cell Patterning and Large Biomolecular Delivery in Mammalian Cells Using Light Pulses.

The recent advancements of single-cell analysis have significantly enhanced the ability to understand cellular physiology when compared to bulk cellular analysis. Here a massively parallel single-cell patterning and very large biomolecular delivery is reported. Micro-pillar polydimethyl siloxane stamp with different diameters (40-100 µm with 1 cm × 1 cm patterning area) is fabricated and then imprint distinct proteins and finally pattern single-cell to small clusters of cells depending on the micro-pillar diameters. The maximum patterning efficiency is achieved 99.7% for SiHa, 96.75% for L929, and 98.6% for MG63 cells, for the 100 µm micro-pillar stamp. For intracellular delivery of biomolecules into the patterned cells, a titanium micro-dish device is aligned on top of the cells and exposed by infrared light pulses. The platform successfully delivers small to very large biomolecules such as PI dyes (668 Da), dextran 3000 Da, siRNA (20-24 bp), and large size enzymes (464 KDa) in SiHa, L929 and MG63 cells. The delivery efficiency for PI dye, Dextran 3000, siRNA, and enzyme for patterned cells are ≈95 ± 3%, 97 ± 1%, 96 ± 1% and 94 ± 3%, with cell viability of 98 ± 1%. Thus, the platform is compact, robust, easy for printing, and potentially applicable for single-cell therapy and diagnostics.

Visible Pulsed Laser-Assisted Selective Killing of Cancer Cells with PVP-Capped Plasmonic Gold Nanostars.

A new generation of nanoscale photosensitizer agents has improved photothermal capabilities, which has increased the impact of photothermal treatments (PTTs) in cancer therapy. Gold nanostars (GNS) are promising for more efficient and less invasive PTTs than gold nanoparticles. However, the combination of GNS and visible pulsed lasers remains unexplored. This article reports the use of a 532 nm nanosecond pulse laser and polyvinylpyrrolidone (PVP)-capped GNS to kill cancer cells with location-specific exposure. Biocompatible GNS were synthesized via a simple method and were characterized under FESEM, UV-visible spectroscopy, XRD analysis, and particle size analysis. GNS were incubated over a layer of cancer cells that were grown in a glass Petri dish. A nanosecond pulsed laser was irradiated on the cell layer, and cell death was verified via propidium iodide (PI) staining. We assessed the effectiveness of single-pulse spot irradiation and multiple-pulse laser scanning irradiation in inducing cell death. Since the site of cell killing can be accurately chosen with a nanosecond pulse laser, this technique will help minimize damage to the cells around the target cells.

Dual-clamped one-pot SERS-based biosensors for rapid and sensitive detection of SARS-CoV-2 using portable Raman spectrometer.

Rapid and sensitive diagnostics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is of utmost importance to control the widespread coronavirus disease 2019 (COVID-19) upsurge. This study demonstrated a novel one-pot surface-enhanced Raman scattering (SERS) based immunoassay to detect SARS-CoV-2, without any washing process using a portable Raman spectrometer. The SERS-immune assay was designed using a regular digital versatile disk (DVD) substrate integrated with Raman reporter labeled silver nanoparticles for double clamping effects. The disks were molded to form nanopillar arrays and coated with silver film to enhance the sensitivity of immunoassay. The SERS platform demonstrated a limit of detection (LoD) up to 50 pg mL-1 for SARS-CoV-2 spike protein and virus-like-particle (VLP) protein in phosphate buffer saline within a turnaround time of 20 mins. Moreover, VLP protein spiked in untreated saliva achieved an LoD of 400 pg mL-1, providing a cycle threshold (Ct) value range of 30-32, closer to reverse transcription-polymerase chain reaction (RT-PCR) results (35-40) and higher than the commercial rapid antigen tests, ranging from 25 to 28. Therefore, the developed one-pot SERS based biosensor exhibited highly sensitive and rapid detection of SARS-CoV-2, which could be a potential point-of-care platform for early and cost-effective diagnosis of the COVID-19 virus.

Metallic micro-ring device for highly efficient large cargo delivery in mammalian cells using infrared light pulses.

Uniform transfection of biomolecules into live cells with high delivery efficiency and cell viability is an immensely important area of biological research and has many biomedical applications. In the present study, we report highly efficient, uniform parallel intracellular delivery of small to very large biomolecules into diverse cell types using a titanium micro-ring (TMR) device activated by infrared (IR) light pulse. A TMR array device (2 cm × 2 cm) consists of a 10 µm outer diameter and 3 µm inner diameter for each micro-ring, and 10 µm interspacing between two micro-rings. Upon IR (1050 nm) pulse laser irradiation on the TMR device, photothermal cavitation bubbles are generated, disrupting the cell plasma membrane, and biomolecules are gently delivered into the cells by a simple diffusion process. This TMR device successfully delivered diverse types of small to very large biomolecules such as propidium iodide (PI; 668.4 Da) dye, dextran (3 kDa), small interfering RNA (13.3 kDa), enhanced green fluorescent protein expression plasmid DNA (6.2 kb), and ß-galactosidase enzyme (465 kDa) into human cervical (SiHa), mouse fibroblast (L929), and mouse neural crest-derived (N2a) cancer cells. For smaller molecules (PI dye), delivery efficiency and cell viability were achieved at ∼96% and ∼97%, respectively, with a laser fluence of 21 mJ cm-2 for 250 pulses. In contrast, ∼85% transfection efficiency and ∼90% cell viability were achieved for plasmid DNA with 45 mJ cm-2 laser fluence for 250 pulses in SiHa cells. Moreover, the intracellular delivery of ß-galactosidase enzyme was confirmed with confocal microscopy and flow cytometry analysis resulting in ∼83% co-staining of ß-galactosidase enzyme and calcein AM. Based on these efficient deliveries of diverse types of biomolecules in different cell types, the device has the potential for cellular diagnostic and therapeutic applications.

Synthesis and characterization of a fluorescent polymeric nano-thermometer: dynamic monitoring of 3D temperature distribution in co-cultured tumor spheroids.

Temperature governs the reactivity of a wide range of biomolecules in the cellular environment dynamically. The complex cellular pathways and molecules in solid tumors substantially produce temperature gradients in the tumor microenvironment (TME). Hence, visualization of these temperature gradients at the cellular level would give physiologically relevant spatio-temporal information about solid tumors. This study used fluorescent polymeric nano-thermometers (FPNTs) to measure the intratumor temperature in co-cultured 3D tumor spheroids. A temperature-sensitive rhodamine-B dye and Pluronic F-127 were conjugated through hydrophobic and hydrophobic interactions and then cross-linked with urea-paraformaldehyde resins to form the FPNTs. The characterization results exhibit monodisperse nanoparticles (166 ± 10 nm) with persistent fluorescence. The FPNTs exhibit a linear response with a wide temperature sensing range (25-100 °C) and are stable toward pH, ionic strength, and oxidative stress. FPNTs were utilized to monitor the temperature gradient in co-cultured 3D tumor spheroids and the temperature difference between the core (34.9 °C) and the periphery (37.8 °C) was 2.9 °C. This investigation demonstrates that the FPNTs have great stability, biocompatibility, and high intensity in a biological medium. The usage of FPNTs as a multifunctional adjuvant may demonstrate the dynamics of the TME and they may be suitable candidates to examine thermoregulation in tumor spheroids.

Functionally gradient three-dimensional graphene foam-based polymeric scaffolds for multilayered tissue regeneration.

Physiological bioengineering of multilayered tissues requires an optimized geometric organization with comparable biomechanics. Currently, polymer-reinforced three-dimensional (3D) graphene foams (GFs) are gaining interest in tissue engineering due to their unique morphology, biocompatibility, and similarity to extracellular matrixes. However, the homogeneous reinforcement of single polymers throughout a GF matrix does not provide tissue-level organization. Therefore, a triple-layered structure is developed in a GF matrix to closely mimic native tissue structures of the periodontium of the teeth. The scaffold aims to overcome the issue of layer separation, which generally occurs in multilayered structures due to the poor integration of various layers. The 3D GF matrix was reinforced with a polycaprolactone (PCL), polyvinyl alcohol (PVA), and PCL-hydroxyapatite (HA) mixture, added sequentially, via spin coating, vacuum, and hot air drying. Later, PVA was dissolved to create a middle layer, mimicking the periodontal fibers, while the layers present on either side resembled cementum and alveolar bone, respectively. Scanning electron microscopy and micro-computed tomography revealed the structure of the scaffold with internal differential porosities. The nanoindentation and tensile testing demonstrated the closeness of mechanical properties to that of native tissues. The biocompatibility was assessed by the MTT assay with MG63 cells (human osteosarcoma cells) exhibiting high adhesion and proliferation rate inside the 3D architecture. Summing up, this scaffold has the potential for enhancing the regeneration of various multilayered tissues.

Combinatorial physical methods for cellular therapy: Towards the future of cellular analysis?

The physical energy activated techniques for cellular delivery and analysis is one of the most rapidly expanding research areas for a variety of biological and biomedical discoveries. These methods, such as electroporation, optoporation, sonoporation, mechanoporation, magnetoporation, etc., have been widely used in delivering different biomolecules into a range of primary and patient-derived cell types. However, the techniques when used individually have had limitations in delivery and co-delivery of diverse biomolecules in various cell types. In recent years, a number of studies have been performed by combining the different membrane disruption techniques, either sequentially or simultaneously, in a single study. The studies, referred to as combinatorial, or hybrid techniques, have demonstrated enhanced transfection, such as efficient macromolecular and gene delivery and co-delivery, at lower delivery parameters and with high cell viability. Such studies can open up new and exciting avenues for understanding the subcellular structure and consequently facilitate the development of novel therapeutic strategies. This review consequently aims at summarising the different developments in hybrid therapeutic techniques. The different methods discussed include mechano-electroporation, electro-sonoporation, magneto-mechanoporation, magnetic nanoparticles enhanced electroporation, and magnetic hyperthermia studies. We discuss the clinical status of the different methods and conclude with a discussion on the future prospects of the combinatorial techniques for cellular therapy and diagnostics.

Automated detection of patterned single-cells within hydrogel using deep learning.

Single-cell analysis has been widely used in various biomedical engineering applications, ranging from cancer diagnostics, and immune response monitoring to drug screening. Single-cell isolation is fundamental for observing single-cell activities and an automatic finding method of accurate and reliable cell detection with few possible human errors is also essential. This paper reports trapping single cells into photo patternable hydrogel microwell arrays and isolating them. Additionally, we present an object detection-based DL algorithm that detects single cells in microwell arrays and predicts the presence of cells in resource-limited environments at the highest possible mAP (mean average precision) of 0.989 with an average inference time of 0.06 s. This algorithm leads to the enhancement of the high-throughput single-cell analysis, establishing high detection precision and reduced experimentation time.

Microfluidic platforms for single neuron analysis.

Single-neuron actions are the basis of brain function, as clinical sequelae, neuronal dysfunction or failure for most of the central nervous system (CNS) diseases and injuries can be identified via tracing single-neurons. The bulk analysis methods tend to miscue critical information by assessing the population-averaged outcomes. However, its primary requisite in neuroscience to analyze single-neurons and to understand dynamic interplay of neurons and their environment. Microfluidic systems enable precise control over nano-to femto-liter volumes via adjusting device geometry, surface characteristics, and flow-dynamics, thus facilitating a well-defined micro-environment with spatio-temporal control for single-neuron analysis. The microfluidic platform not only offers a comprehensive landscape to study brain cell diversity at the level of transcriptome, genome, and/or epigenome of individual cells but also has a substantial role in deciphering complex dynamics of brain development and brain-related disorders. In this review, we highlight recent advances of microfluidic devices for single-neuron analysis, i.e., single-neuron trapping, single-neuron dynamics, single-neuron proteomics, single-neuron transcriptomics, drug delivery at the single-neuron level, single axon guidance, and single-neuron differentiation. Moreover, we also emphasize limitations and future challenges of single-neuron analysis by focusing on key performances of throughput and multiparametric activity analysis on microfluidic platforms.

Microfluidic mechanoporation for cellular delivery and analysis.

Highly efficient intracellular delivery strategies are essential for developing therapeutic, diagnostic, biological, and various biomedical applications. The recent advancement of micro/nanotechnology has focused numerous researches towards developing microfluidic device-based strategies due to the associated high throughput delivery, cost-effectiveness, robustness, and biocompatible nature. The delivery strategies can be carrier-mediated or membrane disruption-based, where membrane disruption methods find popularity due to reduced toxicity, enhanced delivery efficiency, and cell viability. Among all of the membrane disruption techniques, the mechanoporation strategies are advantageous because of no external energy source required for membrane deformation, thereby achieving high delivery efficiencies and increased cell viability into different cell types with negligible toxicity. The past two decades have consequently seen a tremendous boost in mechanoporation-based research for intracellular delivery and cellular analysis. This article provides a brief review of the most recent developments on microfluidic-based mechanoporation strategies such as microinjection, nanoneedle arrays, cell-squeezing, and hydroporation techniques with their working principle, device fabrication, cellular delivery, and analysis. Moreover, a brief discussion of the different mechanoporation strategies integrated with other delivery methods has also been provided. Finally, the advantages, limitations, and future prospects of this technique are discussed compared to other intracellular delivery techniques.

Nanocellulose, a versatile platform: From the delivery of active molecules to tissue engineering applications.

Nanocellulose, a biopolymer, has received wide attention from researchers owing to its superior physicochemical properties, such as high mechanical strength, low density, biodegradability, and biocompatibility. Nanocellulose can be extracted from wide range of sources, including plants, bacteria, and algae. Depending on the extraction process and dimensions (diameter and length), they are categorized into three main types: cellulose nanocrystals (CNCs), cellulose nanofibrils (CNFs), and bacterial nanocellulose (BNC). CNCs are a highly crystalline and needle-like structure, whereas CNFs have both amorphous and crystalline regions in their network. BNC is the purest form of nanocellulose. The nanocellulose properties can be tuned by chemical functionalization, which increases its applicability in biomedical applications. This review highlights the fabrication of different surface-modified nanocellulose to deliver active molecules, such as drugs, proteins, and plasmids. Nanocellulose-mediated delivery of active molecules is profoundly affected by its topographical structure and the interaction between the loaded molecules and nanocellulose. The applications of nanocellulose and its composites in tissue engineering have been discussed. Finally, the review is concluded with further opportunities and challenges in nanocellulose-mediated delivery of active molecules.

Microfluidic nanomaterials: From synthesis to biomedical applications.

Microfluidic platforms gain popularity in biomedical research due to their attractive inherent features, especially in nanomaterials synthesis. This review critically evaluates the current state of the controlled synthesis of nanomaterials using microfluidic devices. We describe nanomaterials' screening in microfluidics, which is very relevant for automating the synthesis process for biomedical applications. We discuss the latest microfluidics trends to achieve noble metal, silica, biopolymer, quantum dots, iron oxide, carbon-based, rare-earth-based, and other nanomaterials with a specific size, composition, surface modification, and morphology required for particular biomedical application. Screening nanomaterials has become an essential tool to synthesize desired nanomaterials using more automated processes with high speed and repeatability, which can't be neglected in today's microfluidic technology. Moreover, we emphasize biomedical applications of nanomaterials, including imaging, targeting, therapy, and sensing. Before clinical use, nanomaterials have to be evaluated under physiological conditions, which is possible in the microfluidic system as it stimulates chemical gradients, fluid flows, and the ability to control microenvironment and partitioning multi-organs. In this review, we emphasize the clinical evaluation of nanomaterials using microfluidics which was not covered by any other reviews. In the future, the growth of new materials or modification in existing materials using microfluidics platforms and applications in a diversity of biomedical fields by utilizing all the features of microfluidic technology is expected.