paper 2
1. Introduction
Advancements have made it possible to miniaturize biochemical protocols into microchannel networking systems
Efficient and cost-effective tool
2 types of microfluidic devices
Passive
No external energy used
All physics and gravity
Usually used for particle manipulation and mixing liquids
Active
External energy used to disturb particles
Acoustic pressure fields, eclectic fields, magnetic fields, thermal fields, and optical fields can manipulate biological or chemical particles and mix fluids
Usually used for particle trapping and sensing
Functional surfaces
Able to detect certain biological structures
DNA, biomarkers, etc.
Applications
Diagnosing of diseases
Cancer and infectious diseases
Possible application
Organ on a chip, tissue engineering, disease modeling
Biosensing technology
Point of care testing, biosensors, and cell manipulations
2. Microfluidics
Microfluidic devices are primarily classified into two groups: passive and active devices.
Passive devices utilize internal forces, diffusion, and secondary flows for fluid mixing and particle manipulation within geometry-dependent limits.
Active devices depend on external energy sources to interact with target particles, overcoming some of the physical restrictions of passive chips.
While some active fields like magnetic forces are limited to specific material properties, others like acoustic and pressure fields offer broader manipulation capabilities.
These microfluidic platforms are highly versatile tools used for diverse biomedical tasks, including disease diagnosis, organ modeling, and cell sorting.
2.1. Passive Microfluidics
The primary applications of passive microfluidic platforms include fluid mixing, particle focusing, separation, sorting, and isolation.
These devices operate independently of external energy sources, relying instead on the inherent dynamics of the fluid and the channel structure.
Geometrical design is the most critical factor influencing the performance and characteristics of passive systems.
Despite being geometry-dependent, passive microfluidics have opened new horizons in biomedical research through implementations like organ-on-a-chip.
Common mechanisms for passive manipulation include microfiltration, inertial and secondary flows, deterministic lateral displacement (DLD), and pinch flow fractionation (PFF).
2.1.1. Inertial Micromixers
Passive micromixers rely on specific channel geometries to perform chemical reactions and biological analyses.
They often utilize "Dean vortices" or secondary flows found in curved microchannels to improve mixing efficiency at low cost.
Chaotic advection is further enhanced by designing uniquely shaped baffles on channel walls to constantly fold and stretch the fluid.
Experimental studies on novel baffle designs have demonstrated mixing indexes as high as 98% even at very low Reynolds numbers (e.g., Re = 20).
These mixers are valued for their simplicity and lack of moving parts compared to dynamic systems.
2.1.2. Sorting, Separation, and Isolation
(a) Microfiltration
This technique uses the size of floating components to trap particles in restrictive elements like membranes, pillars, or weirs.
While dead-end filtration offers higher capture efficiency for large particles, crossflow filtration is often preferred to mitigate clogging issues.
(b) Inertial Focusing and Secondary Flows
In laminar flow, particles move towards equilibrium positions due to the balance of shear-induced lift and wall-induced forces.
Curved channels introduce Dean flow—a secondary flow perpendicular to the primary flow—which adds centrifugal drag to the particles.
(c) Deterministic Lateral Displacement (DLD)
DLD platforms use specific post-array configurations to achieve continuous particle separation at low Reynolds numbers.
Triangular micro-posts have been successfully used to isolate viable circulating tumor cells (CTCs) from blood while reducing the stagnation zones that cause clogging.
(d) Pinch Flow Fractionation (PFF)
PFF involves combining a sample stream and a sheath flow into a narrow "pinched" segment where particles are pushed against a wall.
Separation is achieved because the center of mass for different-sized particles follows different streamlines as the channel widens after the pinched segment.
2.1.3. Droplet Microfluidics
Droplet microfluidics allows for the creation and manipulation of discrete, high-throughput volumes of immiscible liquids like oil and water.
These platforms are essential for applications in microbiology, single-cell analysis, cell culture, and drug delivery.
Common passive channel configurations for droplet generation include crossflow (Y or T-junctions), co-flow, and flow-focusing geometries.
Flow-focusing designs are particularly effective for producing small, spherical droplets with minimal contact with the channel walls.
Droplet size and characteristics can be adjusted by modifying rheological properties, flow rate ratios, and interfacial tension using surfactants.
2.2. Active Microfluidics
Active devices generate fluid streams using external energy to disturb particles or liquids inside the microchannels.
The use of external forces (acoustic, electric, magnetic, thermal, etc.) allows for more precise manipulation of biological and chemical particles.
These systems are highly versatile for disease diagnosis because they can include sensors that transduce biological changes into electrical or optical signals.
Active microfluidics can overcome the inherent geometrical limitations of passive devices by adjusting the external field intensity or frequency.
Functional surfaces coated on the transduction area enable these devices to sense unique biological structures like DNA and biomarkers.
2.2.1. Dynamic Micromixers
(a) Acoustic Field-Driven Micromixers
These mixers use acoustic waves generated by interdigitated transducers (IDTs) to create streams that perturb droplets for rapid mixing.
Surface acoustic wave (SAW)-based transducers are frequently used because they can be fabricated in various geometric shapes to optimize mixing efficiency.
(b) Pulsed Field-Effect Micromixers
Mixing is achieved momentarily through pressure fields generated by an oscillation unit controlled by switching frequencies.
Studies have reported high mixing efficiencies ranging from 75% to 99%, depending on the specific flow rates and oscillation frequencies used.
2.2.2. Particle Separation
Active particle separation is used for critical biomedical tasks such as purifying submicron particles or isolating cancer cells from healthy ones.
Thermal field applications are available but limited to particles that are not susceptible to damage from high-temperature gradients.
Magnetic field gradients created by permanent magnets or electricity-driven coils can separate magnetically sensitive cells like RBCs and WBCs from blood.
Acoustophoresis uses the interaction between acoustic waves and material properties to drive particles towards specific streamlines based on density and size.
Integrated "cascade systems" can combine multiple active separation methods to increase overall purity and efficiency.
2.2.3. Focusing, Sorting, and Enrichment
Focusing phenomena involve accumulating particles into a single point or a straight line using external forces.
Acoustic standing waves created by opposite electrodes generate pressure points that drive particles into specific paths based on their size.
Dielectrophoresis uses electric fields to manipulate particles based on their polarizability relative to the surrounding medium.
Optical field-based transducers can create gradients inside microchannels to focus and sort nanoscale biomolecules.
Enrichment techniques aim to increase the density of specific target particles within a sample for improved detection and analysis.
2.2.4. Particle Trapping
Particle trapping creates "physical tweezers" using acoustic, electric, magnetic, optical, or thermal fields as driving forces.
High-pressure points known as "acoustic tweezers" can trap particles ranging from micron to sub-micron sizes.
Positive and negative dielectrophoresis (pDEP and nDEP) regions are used to trap particles based on the distribution of the applied electric field.
Magnetic particles can be predicted and controlled by manipulating external magnetic energy to drive them toward low-energy regions.
Thermal tweezers utilize Joule heating-induced temperature gradients to drive controllable electric currents for manipulating live cells and microparticles.
3. Fabrication of Microfluidic Devices
Special production techniques required due to resolution constraints and difficulty working at small scales
Will cover three main fabrication categories: molding, 3D printing, and nanofabrication/etching
3.1.1 Replica Molding
Most common fabrication method for biomedical microfluidic devices
Process: use negative photoresist (ex. SU8) patterned on silicon wafer via UV photolithography, pour PDMS over mold, cure it, bond to glass slide with oxygen plasma
SU8 is often used due to high resolution, durability, and high aspect ratios
PDMS is often polymer used because flexible, optically transparent, and biocompatible
Negatives: need cleanroom facility, PDMS can absorb small molecules which can affect cellular responses
3.1.2 Injection Molding
High-throughput, cost efficient, high accuracy
Compatible with a wide range of thermoplastics, few steps
Process: melt thermoplastic, inject into mold cavity, cool and remove
3D print inlays can be used inserted into the mold
Negatives: restricted to thermoplastics, mold fabrication is expensive, low resolution
3.1.3 Hot Embossing
Mold shape is transferred to a thermoplastic/polymer film under heat and pressure in a vacuum
Less internal stress in material compared to injection molding
Used for PMMA devices and is applicable to glass and amorphous materials
Negatives: restricted material options, difficulty fabricating complex structures
3.2.1 Fused Deposition Modeling
Extrusion-based 3D printing by melting a thermoplastic filament, extruding it through a nozzle, and then solidifying
Simple and effective, compatible with diverse materials
Negatives: poor fusion between adjacent layers leads to fragility under compression, also difficult to achieve suitable channel transparency/small channel sizes
3.2.2 Vat POlymerization
Uses UV light to cure photosensitive resin by moving through each layer
SLA (scanning laser) used for microneedle arrays with biocompatible resin for drug delivery
DLP (stationary UV with moving build plate) used in paper-based microfluidic devices for simultaneous multi-biomarker detection via smartphone
Biocompatiblity of commercial photopolymers is currently being studied
3.2.3 Multi-Jet Printing/Polyjet
Highly accurate
Droplets of photosensitive resin ejected from inkjet printhead, then UV cured
Supports multiple materials in a single build
Applications include sub-millifluidic actuators, wearable sweat collection devices, microfluidic valves
3.2.4 Two Photon Polymerization
Focused laser cures only the nonlinearly excited focal spot in a liquid resin, this enables nano-scale features
Highest resolution among 3D printing methods
Applications include biomimetic placental barrier structures, microneedle arrays, transparent fused silica glass microstructures, coaxial lamination mixers
3.3.1 Nanofabrication
Standard photolithography is insufficient for nanoscale features due to long UV wavelengths
EUV lithography uses 13nm wavelength and is very high resolution
Electron beam lithography (EBL): high energy electron beam exposes resist, also very high resolution
Both EUV and EBL have high costs and limited throughput and are not widely used in nanofluidics
Nanoimprint lithography: mechanical pressing of a mold into resist, then hardening (by thermal/chemical/optical), widely used, enabled creation of polymer nanostructure chips for cancer cell detection
3.3.2 Wet and Dry Etching
Used primarily for silicon and glass devices
Wet etching: fast but uses harsh chemicals (HF), produces isotropic/rounded channel profiles, some safety/environmental concerns
Dry etching: slower but precise and controllable, products anisotropic/straight profiles
Both wet and dry etching used in microfluidic devices and biological detection systems
4.1.1 Cancer Detection
Conventional cancer detection (via PET/MRI/CT) involves high radiation/chemotherapy exposure, microfluidics offers minimally invasive alternatives
Theranostic nanoparticles combine drug delivery and imaging, allows monitoring of drug release, cancer state, and efficacy
Microfluidic systems model cancer mechanisms like apoptosis, drug resistance, invasion, and metastasis
Multi-organ chips can mimic lung cancer metastasis, electrical impedance systems track single cell migration
Circulating tumor cell (CTC) isolation uses antibody based CTC chips
4.1.2 Cardiovascular Disease Detection
CVD are leading cause of premature death, diagnosis requires accurate, fast, and low cost tools
Key bloodborne biomarker include cardiac troponin I, fibrinogen, C-reactive protein, NT-proBNP
Microfluidic platforms offer portability, fast analysis, and low reagent consumption
4.1.3 Respiratory Infection Detection
Microfluidic detection strategies classified as antigen detection, antibody detection, or nucleic acid detection
Digital microfluidic qPCR cartridge detects covid gene with uniform droplet formation adn temperature control
Paper based devices, used smartphone detection from saliva via immunoagglutination and capillary flow, airborne droplet capture directly onto paper chip
4.2 Drug Discovery and Delivery
Traditional drug delivery causes high toxicity and side effects, microfluidics enables controlled targeted delivery
Drug carriers: dendrimers, micelles, liposomes, polymers, metallic nanostructures (all must be biodegradable, biocompatible, and stimuli-responsive
FDA approved examples include Doxil, inFed, Abraxane, onpattro and comirnaty
Microfluidic particle fabrication methods include droplet/flow lithography, electrohydrodynamic cospraying, soft lithography, micromolding
Particle geometry affects biodistribution - ellipsoids/nanorods adhere better to vessels than spheres, inhalation bypasses extravasation, oral polymeric nanoparticles show enhanced GI activity
Microfluidics allows personalized therapy via adjustable dosing and combined drug strategies
Organ on a chip and body on chip platforms used for preclinical testing of barriers
Applications also include gene therapy and gene editing delivery systems
4.3 Disease Modeling
2D cell culture has significant limitations such as altered cell morphology, disrupted cell-ECM communication, unrestricted nutrient access that is not physiologically representative
3D platforms better mimic in vivo tumor microenvironments and improve drug response similarity to animal models via increased cell to cell adhesion
Disease on a chip models allow control of shear force, cell patterning, cell to cell communication and biochemical gradients
4.3.1 Cancer Modeling
Focus areas: invasion, intravasation, extravasation, and tumor microenvironment modeling
Tumor immune microenvironment chip models neutrophil role in ovarian tumor invasion
Detachable PDMS gradient generator allows for pump-free characterization of chemotactic factor transmission through a hydrogel
3D breast cancer invasion chip compared MDA-MB-231, MCF-7, and CAMA-1 invasion behavior
Intravasation modeling: TRPM7 identified as fluid shear sensor regulating cancer cell intravasation
Combined invasion and intravasation chip incorporated 3D tumor, stroma, and vasculogenesis in a single device
L-TumorChip: three layer platform combining tumor stroma and microvasulature to test stromal and drug response effects
Colorectal cancer on a chip includes CRC facets, stromal cross-talk, and mechanical force
Pancreatic cancer organoid chip uses patient-derived organoids with epithelium-stroma communication
4.3.2 Neurological Disease Modeling
Microfluidic CNS models look at axons, synapses, neuronal networks for neurological diseases
Huntington’s disease corticostriatal network chip revealed the importance of pre-synaptic compartment in early HD
ALS model using iPSC-derived skeletal muscle and motor neurons evaluated muscle contraction and motor neuron viability
Peripheral nervous system demyelination chip modeled myelination, demyelination, remyelination chip with coculture of motor neurons and Schwann cells sustained over 40 days
ALS FUS mutation chip showed poor neurite regeneration
4.3.3 Pulmonary/Lung Disease Modeling
Lung on a chip models evaluate drug toxicity under physiological conditions, used for drug screening and personalized therapy
Pulmonary edema model exposed alveolar-capillary interface with human endothelial and epithelial cells to fluid flow and cyclic mechanical strain to simulate breathing
Pulmonary arterial hypertension chip models molecular and functional alterations in vascular cells
COPD chip quantifies neutrophil chemotaxis in sputum from COPD patients, uses neutrophil migration as diagnostic marker
4.3.4 Liver Disease Modeling
Liver disease progresses silently, so in vitro microfluidic models are important for understanding pathogenesis
NAFLD on a chip - an inverted pyramid microwell co-culturing HepG2 and HUVECs models steatosis progression
Alcoholic liver disease chip exposes mono and co cultured spheroids to ethanol and evaluates CYP450 activity and hepatic function at different injury levels
4.4 Tissue Engineering
Goal to regenerate bioengineered tissues using biocompatible scaffolds that mimic ECM, support cell proliferation, and biodegrade without toxicity
Static 2D culture limits cell to cell and cell to ECM interactions, microfluidics introduces continuous flow and controlled microenvironments that better replicate in vivo conditions
MIcro perfusion systems deliver nutrients and remove waste continuously through small channels
4.4.1 Replication of the Cellular MIcroenvironment
Cellular microenvironment = dynamic interactions of cells, interstitial fluid, and ECM
Interstitial flow (IF) = one-way fluid transport through ECM, affects cancer metastasis and soluble factor transport through tumor stroma
This is modeled on a chip
Cell migration altered by microenvironment stiffness, can vary stiffness and dimensionality of the chip to cause cell separation
4.4.2 Fabrication of Biomaterials
Microfluidics is cost effective and manageable for producing nanoparticles, microfibers, and microspheres
Magnetic chitosan microspheres (MCMs) = microfluidic fabrication with a uniform size, promotes wound healing, angiogenesis, and antibacterial activity
Collagen microfibers are produced by fragmentation with continuous flow and shear stress alteration
4.5 Organ on a chip
A microfluidic device that is used to replicate human tissues
It’s used for investigating how pathophysiology of diseases and therapeutic approaches
Uses microfluidic channels as well as engineered tissue
Replicates the in vivo cell microenvironments communications better than two dimensional cell cultures
Is used as an ethical and more reliable replacement for two dimensional cell cultures
Has mainly been used for replicating the Liver, heart,gut,kidney,lung and brain
4.5.1 Gut-on-a-chip (GoC)
Used for studying the gut triggers for diseases
Three dimensional models are the only model that can replicate the gut microenvironment
The chip has a Intestinal epithelial channel, ecm coated membrane, a vascular endothelial chanel, and a porous membrane
Gut on a chip can replicate the gut dynamics because of the perfused microchannels in the chip, and the intestinal cells in the chip
The channels mimic the invivo morphology of the gut
In the example they used this technology to understand microbial interactions with gut microbiota
In this example the chip was used to explore pathogenic diseases
When used for testing drug absorption, they use tissue cultures which helps give a more accurate reading for the chip, but also makes the chip have a shorter shelf life
Intestinal explant barrier chips are used to analyze intestinal permeability.
Intestinal explant barrier chips used humine and porcine intestinal tissue in different channels to test the intestinal absorption
4.5.2 Bone on a chip
Conceicau et al wanted to test the cellular interaction of breast cancer, in this they found out that the signaling of breast cancer increased with the release of pancreatic signaling
In Conceicau et al they had osteoclast, breast cancer cells and neurons all cultured in different chambers of the device.
The different channels allow for signaling to happen.
Glaser created a microfluidic device that allowed for two bone marrow samples (endosteal and perivascular) separated by two channels
All the studies created a microfluidic device with multiple channels to allow for cell signaling
4.5.3 Liver on a chip
Similar to the chips before they wanted to test fatty liver disease and created a microfluidic device with dual blood samples from the hepatic artery and the hepatic portal vein.
Leung et al used chips were modeled after the adipose tissue
In the chip they use free fatty acid to trigger lipid formation in adipose tissue cells, this is used for studying obesity
Lee et al created gut liver chips to test hepatic steatosis
In this chip they have two layers, a gut reservoir and liver reservoir connected by channels
Heart liver chips are used to monitor the damage on heart cells more efficiently then static cultures
Heart liver chips can help investigate cardiotoxicity in the heart
Similar to the other organ chips, they use multiple chambers and channels to test signaling in the cell
4.5.4 Brain on a chip
Brain on a chip is being used to study epileptic seizures
They created a microfluidic device model with a microelectrode array that detects seizure-like activity.
Additionally human pluripotent stem cells and differential neurons were added to a channel, and then Kanic acid was added to induce the seizure activity which the microelectrode array detects
Different then the chips before the brain on a chip uses a chemical stimulant to create an electric response
They created a GBA chip to test blood and brain communication, two channels one with gut endothelial cells and one with brain endothelial cells
In the chip they added Lipopolysaccharide to the gut barrier
This induced inflammation in the gut barrier which influenced a response in the permeability of the Blood brain barrier
4.5.5 Heart on a chip
The key elements for heart on a chip platforms are Human induced pluripotent stem cells and differentiated cardiomyocytes
Unfortunately Human induced pluripotent stem cells are usually immature making this process hard to replicate
Zhang et al created a long term dynamic culture of Human induced pluripotent stem cells, which they then applied electrical stimulation to mature the cardiomyocytes
The heart on a chip can be used to evaluate drug efficiency and cardiotoxicity
4.5.6 Kidney on a chip
They want to mimic glomerulus to investigate kidney physiology and diseases
They created a chip that reproduced the glomerulus barrier utilizing hiPSCs nephron progenitor cells and vascular endothelial cells separated into different inlet channels but connected by a main chamber allowing for signaling
The big thing to note is that they use personalized cells for the device and not the broader cells that they use for every other chip
4.5.7 Lung on a chip
They are reproducing the air blood barrier
The chip they created for this is biodegradable and has a elasticity of biological membrane due to the presence of collagen elastin and other proteins in the lung.
The chip actually recreates the mechanical aspect of breathing
4.6 Microfluidic biosensors
Biosensors provide us with rapid analysis
They are categorized by four groups enzymes based, nanozymes based, antibodies based and nucleic acid based
Important advantage/disadvantage table
4.6.1 Enzyme based microfluidic devices
Usually uses redox enzymes
They are perfect because electrochemical monitoring can detect the reactions
They also are being used because they have a high selectivity,biocatalytic activity, and precise enzyme substrate interactions
They are coupled with microfluidic devices because of automation, small and stable sensing area, and multiplexed functions
There issues are reliable, have long term stability and reusability
Enzyme immobilization are used to address these issues
Enzyme immobilization puts the enzyme onto the surface of the transducer as well as on the micro channels.
Enzyme immobilization usually relies on adsorption,covalent bonds,crosslinking, and entrapment of enzymes
Enzyme immobilization uses bonding to immobilize the enzyme without disrupting the essential structure of the enzyme
Covalent bonding is the hardest of the three bonding types, it requires strong interactions between surface groups of the enzyme and the surface or between two enzymes
Crosslinking forms three dimensional enzymes by using it for immobilization
Enzyme can be encapsulated in either organic or inorganic polymers
Usually when they are encapsulated in polymer they are used for glucose level measurements (diabetes studies)
The enzyme used for this is Glucose oxidase, which is easy to get low cost and is durable against ph and temperature
Sun et al created a device, they use glucose oxidase electrode substrate and a microchannel made of high concentration buffer loaded with kimwipes to get a distinctive stable glucose measurement
They use the high concentration buffers ability to maintain its same ph when they flow into the channel to get a accurate glucose measurement
The device measures noninvasive biofluids (tears, sweat, saliva)
Shitanda et al used Lactate oxidase covalently bonded to MgO electrode with the help of a glycidyl methacrylate, which was added to eight sweat collecting channels to monitor the level of another biomarker
Tzianni et al created a smartphone coupled sim card biosensor to monitor creatinine measurement
They added the creatinine deiminase enzyme onto a ph responsive copolymers which demonstrated three conductive electrode configurations
This was then mounted onto a sim card
A enzyme based paper microfluidic system ( μPADS) allow for a cellulose matriz which is flexible, thin, and cost effective
They created a microfluidic device that can use low test alcohol, glucose and lactate levels in low volumes of sweat
They can monitor free amino acids in blood to give information about the state of several diseases
They created a device that uses aminoacyl-tRNA synthetase to analyse histidine,tryptophan,glycine and lysine levels
In this microfluidic device their was four detection zones for each amino acid type with detections zones that all had aminoacyl-tRNA synthetase
Another device uses sweat to measure the amount of ethanol and ammonia
In a multi layer device they use polymer pumps and capillary burst valves to mix the enzyme with the sweat
4.6.2 Nanoenzymes
Nanoenzymes are similar to enzymes, but they are easier and less expensive to produce, they have a longer storage time, and can withstand harsh conditions( high ph)
They are used similarly to regular enzymes, but just more stable and cheaper
4.6.3 Antibodies
They are used because immunogen can be detected with no prior purification steps
They can use fab fragments instead of the whole antibody which improves precision
Was used in covid
Electrode detectors are coated with gold, fluorine doped tin oxide, graphene, etc which will indicate if there is a antigen/antibody interaction
Dna microfluidic detection devices use dna fragments followed by dna hybridization of immobilized complementary target sequence
Traditional methods of dna detection have two distinct steps amplification and then base pairing on a gel substrate
The detection works by receiving relevant change in physiochemical signals
There are three biosensors to detect nucleic acid, PCR, Crispr, and isothermal amplification
In Isothermal amplification the thermocycler is replaced by an incubator or water bath integrated device (Stays at the same temperature)
PCR, 1 denature the dna to separate the double strand, 2 primers attach to the target, and then a new dna strand is built
The isothermal amplification devices discussed are Recombinase polymerase amplification, loop mediated isothermal amplification, which all are efficient and accurate
Crispr guides the rna to the device to bind to a target dna and cleave it, this is added to a microfluidic device with dna reporter and a cas12-gRNA. It can detect the target nucleic acid within less than 35 min
4.7 Artificial cells
They use double emulsion droplets to model mechanosensitive artificial cells
The device is used to trap mixed drops in the chamber which increases the pressure due to cell activity
4.8 Cryopreservation
This technique is prone to cell damage, osmotic shock and ice crystals
It can rapidly cool biological materials for long term storage
Ozylu et al created a chip to preserve cells on sensor surfaces by cryopreserving the cells with modified polyethylene vinyl acetate to make the sensor durable to the high temp change and then fast thawing the sensor to decrease thermal mass and preserving the cells
4.9 applications
All the topics that were explained before
These devices can be used for diagnosis, therapeutics, organ modeling
aThey can be used to detect diseases, drug discovery, disease modeling, organ on a chip, tissue engineering