Hair and Nail-On-Chip for Bioinspired Microfluidic Device Fabrication and Biomarker Detection

Figures & data

Figure 1. Hair and nails for the construction of microfluidic organ-on-a-chip platforms and their subsequent utilization in a range of biomedical applications.

Table 1. Hair or nail-fabricated micro/nanofluidic devices.

Sl. No:	Hair/Nail Type	Method of Fabrication	Device Description	Proposed/Possible Applications	Advantages	Disadvantages	Ref
Fabrication of Microfluidic Devices using natural hair/nail
1.	Human/Cat Hair Samples	Soft lithography technique involving the cooling/curing of a degassed PDMS prepolymer over hair pieces fixed on a glass surface to act as templates for creating the microfluidic channels.	PDMS (polydimethylsiloxane)-based microfluidic channels inspired from mammalian hair.	Immunoassays, cell analysis, silk-based microwire synthesis and other potential biomedical applications.	Needs ambient temperatures. No involvement of harsh chemicals. Tunable width and length of microchannels.	Difficult to replicate the microchannel structure on the synthesized microwires.	^[Citation67]
2..	Human Hair	Electrospinning technique : Keratin protein extracted from human hair was mixed with poly(vinyl) alcohol, with glyoxal acting as the crosslinker to form nanofibers upon ejection at a high voltage	Human hair-based nanocomposite fibers with excellent mechanical and antibacterial properties.	Numerous future biomedical applications.	Keratin is a widely available protein with a fibrous structure having excellent mechanical strength.	Challenges in the extraction of keratin owing to the protein’s insolubility in polar solvents.	^[Citation79]
3.	Human Nails	Soft lithography for curing PDMS into two blocks, followed by sandwiching a nail plate between them and sealing using oxygen plasma treatment. Pipette tips were inserted into holes punched in the blocks to act as reservoirs	A simple micro/nanofluidic device having a perm-selective membrane fabricated from nano-porous structures, such as human nails.	Various pharmaceutical applications – Point-of-care diagnostics Drug Development	Human nails serve as easily available raw material. The resulting device is biodegradable. Portability owing to its small size.	Possibility of substantial errors due to contamination of the raw material (nail plates). High perishability of the device limits its life-span.	^[Citation69]
4.	Artificial Cilia	Controlled patterning of piezoelectric sensors on silicon oxide layers, followed by photolithographically depositing a layer of gold to define a cantilever beam. Plastic deformation magnetic assembly : Applying a magnetic field to rotate off the cantilever beam from the substrate at a desired angle	Artificial hair cell sensors consisting of a cantilever beam and utilizing a silicon substrate for support.	Numerous biological applications, such as hearing and flow sensing.	Straightforward and sturdy functioning of the device. Increased yield in the sensors by the improved PDMA process.	Multiple substrate and sacrificial layers are necessary to be incorporated. Problem of having a mechanical slack in the hinge structures. Tedious process of optimizing the electrical connections in the device.	^[Citation89]
5.	Bio-mimetic silicone cilia	Designing of biomimetic artificial cilia by – Silicon mold fabrication using deep reactive ion etching (DRIE) on a photoresist patterned on silicon substrate Soft lithography to cure PDMS into ciliary structures on the designed silicon wafer Assembly of the microfluidic device using an underwater fabrication method.	Highly compliant artificial cilia capable of being actuated by a piezo-actuator to mimic the beating of biological cilia.	Development of micromixers and micropumps for the manipulation of fluid flow.	Avoids the collapse of cilia during functioning. Resulting actuation frequency falls in the range of the beating frequency of biological cilia.	–	^[Citation73]
6.	Synthetic Cilia	By utilizing computational modeling, a microfluidic channel was lined with non-motile, elastic cilia. This channel experienced a pressure driven fluid flow, followed by simulations of the same with non-motile, elastic cilia using computational modeling.	A hybrid computational model consisting of two numerical methods - LBM (Lattice Boltzmann Model) and LSM (Lattice Spring Model) for simulations of pressure-driven fluid flow in a microchannel lined with elastic cilia.	Hydrodynamic separation studies. Trapping and most importantly, the filtration of microscopic biological particles such as cancer cells for biomedical and research purposes.	Effortless and straightforward protocol for the synthesis of the cilia fibers.	Lack of indication of any direct practical applications through modeling and simulations alone.	^[Citation95]
7.	Magnetic artificial Cilia	In-house fabrication method involving rotating of a roll consisting of PDMS-based micropillars using an electromagnet on a substrate coated with a precursor film, resulting in the pulling out of filaments to function as fine ciliary structures	Magnetically actuated artificial cilia with super paramagnetic beads as backbones.	Detection of chemicals/biomolecules in the environment/ in a miniaturized biosensor system. Introducing self-cleaning property to surfaces or self-assembly based on surface tension patterning.	Reduced cost and time for device fabrication. Potential to “plant” stable artificial cilia into existing microfluidic channels. Flexibility and structural stability in the cilia. Possibility of in-situ fabrication of artificial cilia in existing microfluidic devices.	Highly tedious and costly process for the production of real-life products. Requires micro-system techniques such as photolithography. Low reproducibility due to lack of control in fabrication. Labor-intensive and time-consuming fabrication protocols.	^[Citation93]
8.	Artificial cilia or hair-like structures	Creation of iron nanowires by electrodeposition on a nano-porous aluminum oxide layer, followed by mixing with PDMS to create a magnetic nanocomposite. Mounting of the composite on a glass substrate, with eventual curing of a PMMA mold to fabricate fluidic channels to act as GMI sensors	Nanocomposite pillars consisting of magnetic iron nanowires and PDMS polymer integrated with a GMI flow sensor for mimicking natural cilia.	Flow and Vibration sensing in both air and water.	Desirable properties, such as chemical resistance, low Young’s modulus value and high elasticity of PDMS. Simple and robust fabrication protocol of GMI sensor.	Emerging magnetic field sensors, such as magnetic tunnel junctions might already be more advantageous than the given GMI sensor.	^[Citation98]
9.	Artificial Cilia	Two-colour lithography-based method: a) First illumination at 365 nm of ultraviolet light for generating anchor strips on a layer of poly(N,N-dimethylacrylamide) b) Second illumination at less than 300 nm of ultraviolet light to generate magnetic cilia	Artificial cilia integrated into a microfluidic channel capable of actuation by a permanently rotating magnet under water.	Construction of magnetic actuators in microfluidic systems for sensing and manipulating fluid flow.	Flexibility of incorporating cilia of various physical dimensions in the device. Magnetic actuation serves as the most effortless method of controlling ciliary movement.	Requires a significant source of illumination to operate. Tedious and expensive procedure of fabrication of artificial cilia.	^[Citation88]
10.	Magnetic artificial, elastic cilia	Fabrication of flexible filaments using a two-step lithographic process in conjunction with the deposition of a magnetic nickel–iron permalloy.	Metallic thin-film cilia with tunable physical characteristics capable of asymmetric motion like biological cilia upon magnetic actuation.	Microfluidic pumping, mixing and other fluid handling processes. Biomedical assays for handling and analyzing biological samples.	No interference with biological systems	Involves complex chemistry to realize the lithographic step	^[Citation71]
11.	Human follicular unit extracts (FUE)	Creation of a miniaturized PDMS block containing the required channels, micropumps and openings for culture compartments using soft lithography and replica molding, followed by permanent fixing on a glass slide.	Dynamically perfused chip-based bioreactor for enhancing the maintenance of ex-vivo skin and hair follicles	Short and mid-term cultures of ex-vivo tissues. Potential long-term organotypic functional models. Possible manipulation of cell-cell communication and microenvironment by dynamic perfusion.	Prolonged culture of in-vitro skin equivalents (SEs). Control over extracellular and pericellular diffusion gradients, and distances traveled by signaling molecules using dynamic perfusion	Complexity and heterogenic nature of the native skin tissue yet to be considered in future studies. Partial signs of regression phase observed in the cultured FUEs.	^[Citation100]
12.	Synthetic Cilia	Fabrication of a programmable microfluidic device consisting of uniformly distributed cilia on the bottom of a microfluidic channel, followed by their actuation to study its interaction with particles using simulations.	A 3-D simulation-based model coupling the Immersed Boundary method and Lattice Boltzmann Fluid Model for studying the capabilities of actuated cilia of separating particles based on their physical properties.	Prediction of cilia-particle interactions using 3-D simulations can be highly useful in applications, such as cell isolation and tissue engineering.	Nondestructive method for studying cilia-particle interactions. Accurate predictions possible for practical biomedical applications.	Actual experiments still required to analyze cilia-particle interactions.	^[Citation101]
13.	Carbon Nanotube Fibers (“hair-like” conductive microwires)	Particle-coagulation spinning (PCS) process – Spinning of fibers in dispersions of single-walled carbon nanotubes (SWNT), followed by ejection through a needle into a coagulation bath for the production of carbon nanofibers in the presence of poly(vinyl) alcohol	–	Possible utilization in the design of supercapacitors, electrochemical transducers, artificial muscles, and microwires. Designing of permeable conduits in micro- and nanofluidic devices and media packaging material.	Nontoxic in nature. Support attachment, spreading and growth of mammalian cells. Permissive to neural cell extension.	Long-term exposure elicites significant, acute, and cytotoxic effects.	^[Citation72]

Figure 2. Schematic illustration of the pioneering fabrication of a microfluidic platform from mammalian hairs and its subsequent application in microwire synthesis, immunoassays, cell sorting and manipulations. Adapted with permission from Ref. [67].

Figure 3. Diagrammatic depiction of the process of designing a nanofluidic device from a human nail plate, succeeded by its detailed characterization to demonstrate nanofluidic phenomena, such as Perm-selectivity. Adapted from Ref. [69].

Figure 4. Simplified demonstration of a novel two-color lithography process of constructing arrays of magnetically actuated artificial cilia with desired geometrical and mechanical properties. Adapted with permission from Ref. [88].

Figure 5. Fabrication scheme of a dynamically perfused chip-based bioreactor capable of prolonging the maintenance and testing periods of ex-vivo human skin and follicular extracts for a range of biomedical applications. Adapted with permission from Ref. [100].

Figure 6. Microfluidic hair and nail-on-a-chip devices as noninvasive and reliable alternatives to conventional sampling in a variety of diagnostic applications.

Table 2. Hair/nails as biomarkers in microfluidic assays.

Sl. No	Source of Sample	Detection Method	Dynamic Range	Limit of Detection (LOD)	Analysis Time	Advantages	Disadvantages	Ref
Detection of Chemicals from Non-Human Hair Samples
1.	Hair samples from Bovine	Pioneering development of a microfluidic immunosensor on a gold nanoparticle-modified (AuNP) screen-printed carbon electrode for the sensitive detection of clenbuterol. The microchannels and designs were made on a miniature PMMA substrate (polymethylmethacrylate) by direct machining using laser scribers	0.01 to 1000 (ng/mL)	0.008 ng/mL	–	Highly useful for rapid, sensitive and selective detection in an “in vitro” fashion. Potential for portability and automation for food safety analysis	–	^[Citation111]
2.	Hair samples from swine	SERS-immunoassay-based detection on a switch-on-chip multilayered paper-based microfluidic analytical device (μPAD) for the detection of clenbuterol (CL)	0.1–100 pg/mL	0.1 pg/mL	2 h for immunoassay	Control over the timing of the reaction due to switch mechanism. Cost-effectiveness and accuracy in results facilitates point-of-care testing in real samples.	Antibody amount to be comparable to that of CL for generating strong signal. Immunoassay involves inherent washing step. Too large analysis time for point-of-care testing
3.	Hair samples from swine	SERS-immunoassay-based detection on a switch-on-chip multilayered paper-based microfluidic analytical device (μPAD) for the multiplexed detection of clenbuterol, terbutaline, and ractopamine. Device design : Microfluidic sensor having filtration and detection regions pattern on a paper substrate using wax printing: direct drawing using a wax pen; using an inkjet printer; direct printing of pattern on filter paper using a wax printer	10 to 150 ppb (ng/g)	20, 20, and 30 (ng/mL) for clenbuterol, ractopamine and salbutamol, respectively.	Electrodeposition time in a range of 10 –80 s.	Label-free detection. No requirement of sample purification or separation.	–	^[Citation114]
4.	Hair samples from swine	A novel µPAD sensor fabricated using a wax printing method for the simple and rapid quantitative measurement of β-agonists in swine hair samples.	4.0 × 10^−8 to 1.0 × 10^−5 (mol/L)	2.0 × 10^−8 (mol/L)	–	Capable of miniaturization for on-site detection of beta-agonists in point-of-care scenarios. The device is capable of rapid, convenient and cheap functioning of.	Methods, such as high-performance liquid chromatography, gas chromatography, capillary electrophoresis and surface-enhanced Raman spectroscopy have better sensitivity than the given device.	^[Citation115]
5.	Swine hair samples from three different sources	An assay-based platform integrating -MIA (Micro Flow Injection Analysis) and chemiluminescence assembled on a two-layered borosilicate microfluidic chip to detect the presence of β-antagonists. Fabrication scheme: Microchannel design formed on a glass substrate using photolithography; etching done on exposed glass surface to create microchannels; creation of hole inlets and outlets using a bench drilling machine for introduction of fluids; joining of top and base glass layers by controlled melting and cooling procedures.	–	2.0 × 10^−8mol/L of ractopamine, 1.0 × 10^−8mol/L of terbutaline and 5.0 × 10^−7mol/L of salbutamol.	< 45 s	Portability and rapidity of analysis. Minimal sample consumption ensures by the compact design of the device.	High sample preparation time. Sample preparation only feasible in an alkaline medium. Need of elevated temperatures for device fabrication.	^[Citation138]
Using Hair Samples as DNA Samples
6.	DNA from telogenic hair and hair shafts	Commercially available microchip capillary electrophoresis (MCE) for analysis of mitochondrial (mt) DNA from hair samples Design Overview – Sodalime glass microchips with 15 mm long channels for sample loading and analysis	30–60 copies of mtDNA template after PCR (polymerase chain reaction) amplification	500 mtDNA copies of HVR1 template sequence for sequencing	120 seconds per run of electrophoresis	Robust sequence data due to normalization by laser-induced fluorescence (LIF) Rapid assay to differentiate between human and non-human samples.	Multiple steps involving DNA extraction, PCR and MCE analysis. Further optimization needed in the electrophoretic conditions and sieving matrix.	^[Citation106]
7.	Human hair roots and hair follicles, E.coli samples	Designing of channels on PMMA substrate using a computer numeric controlled milling machine. Through-hole creation in the inlet and outlet ports of substrate using drilling, followed by inserting silicon tubes into the holes for fluid introduction.	–	42–45 CFU/mL of E.coli bacteria	∼102 minutes (sample preparation and PCR amplification)	Single-step operation comprising sample preparation. Device compatibility with all the operation steps. System overcomes limitations of Chelex as a solid-phase resin.	Band Intensity less than that of an off-chip PCR assay. Improvements needed in design for point-of-care applications with minimum user-defined steps.	^[Citation107]
8.	DNA samples from human hair.	A hybrid system comprising PCR (Polymerase Chain Reaction) & MCE (Microchip Electrophoresis) to ascertain the presence of miniSTRs (mini-short tandem repeat) in forensic and evolutionary applications. Four microchips with double T-injection were included in the device consisting of a rectangular microchannel for detection of DNA.	–	–	< 100 seconds	Rapid and sensitive testing. Suitable even for the analysis of deteriorated samples. Amplification efficiency is enhanced by the use of two annealing temperatures.	Multiple steps make the PCR protocol tedious and time-consuming. The system works in an extremely narrow temperature range.	^[Citation121]
9.	Carnivorous Animal Hair	Microfluidic assay for performing noninvasive SNP (single nucleotide polymorphism) genotyping in forensic and evolutionary studies. Array design – Dynamic array having 48 sample and assay inlets each is mounted on a plastic plate to produce 2304 reaction chambers. An integrated fluid circuit (IFC) exists, consisting of valves that deflect under pressure to form a tight seal for controlling fluid flow.	–	–	–	Noninvasive sample extraction. The coherence in the mutation codes for SNP nullifies the need for evaluating data from various sources.	Multiple steps make the protocol tedious to perform. Requires highly stringent operating conditions.	^[Citation123]
10.	Human nail and hair samples	A bioassay combining a novel centrifugal disk for performing DNA extraction from hair samples, and its subsequent detection using loop-mediated isothermal amplification (LAMP) for forensic sex-typing studies.	–	–	30 min	Suitable for point-of-care forensic analysis. Up to thirty sample extractions and purification processes can be simultaneously performed on a single device.	The sex-typing using LAMP involves multiple steps and requires the use of a large number of reagents.	^[Citation122]
Determination of Drugs/Metabolites from Human Hair
11.	Scalp hair specimens from humans	An automated method involving micro-pulverized sample extraction and microchip-based competitive ELISA for the full-range detection of methamphetamine (MA)	0.2–5 ng/mg in neat extracts, 5–250 ng/mg in diluted extracts	̲˂ 0.2 ng/mg of MA	˂ 30 minutes	Automated functioning of the device. Small size and portability for analysis in remote settings. Contamination-free setup without high power requirement.	Antibody-coated beads require replenishment before every new assay. Pulverized extraction still involves steps that impede point-of-care testing.	^[Citation125]
12.	Human Hair	Microfluidic chip consisting of a capillary pump and a nanoflow pump with a degasser, along with a thermostate microwell-plate sampler. The chip was inserted into a HPLC-Chip cube interface, followed by direct mounting on a mass spectroscopy source.	–	0.1 to 0.75 pg/mg	15 minutes	Rapid and sensitive analyte detection. Minimal sample quantity and preparation time required. Compactness and portability offer flexibility for POC applications.	A minute and consistent flow rate must be maintained, which is difficult to achieve. Multiple parameters to be determined before precise identification of the analyte.	^[Citation102]
13.	Human hair samples.	Patent: Microfluidic chip with a SERS-based detection mechanism for quantitative detection of amphetamine chloride.	10^−10-10^−8mol/L	10^−8mol/L	–	Noninvasive extraction and easy storage of hair samples. Portability and compactness for POC analysis. Shows high selectivity and sensitivity in the analysis of actual hair samples.	–	^[Citation126]
Analysis of Biomarkers from Nail Samples
14.	Human Nails	Creation of chip columns on a polyimide film by implementing laser ablation, followed by depositing a noble metal electrode at one end of the channel to apply voltage. Introduction of a column to perform chromatographic separation by coupling to nanofluidic pumping system.	–	(1–10) peptides detected from human nails	> 20 minutes	Integration of multiple steps into a single microfluidic platform allows rapid analysis of complex biological samples. Offers high sensitivity of analysis.	Involves a long sample preparation process. Not suitable for ready-made analysis of samples in resource-limited settings. May not be affordable by certain populations of potential users.	^[Citation137]
15.	Human Nails	A nano-flow chip LC (liquid chromatography) coupled with Q-TOF-MS/MS (quadrupole time-of-flight tandem mass spectrometry) for the simultaneous determination of nine polyamines from human nail samples.	–	0.5–5 fmol and 0.01–0.1 fmol using MS and MS/MS, respectively.	18 minutes	Noninvasive and straightforward procedure. The nano-flow chip yields perfect separation of the targeted analytes. The Q-TOF-MS/MS ensures accuracy and precision in sample analysis.	The highly polar and hydrophilic nature of nails makes them tedious to work with. A more precise and rapid sample preparation technique is required.	^[Citation130]
Detection of Physical Quantities from Hair/Nail Samples
16.	Microstructures constructed from the conducting PEDOT (poly (3,4 ethylene dioxythiophene:poly (styrene sulfonate)) polymer.	Printed PEDOT microstructures inspired from the flow sensing nature of hairs in arthropods.	Air flow velocity in the range of 0.66 m/s–0.97 m/s	0.66 m/s of air flow velocity	5 min	The PEDOT: PSS micro-hairs are flexible and elastic in nature, thereby regaining their original shape after air flow velocity determination.	Involves the use of expensive equipment. Comprises of multiple time-consuming steps that require great precaution to be followed.	^[Citation74]
17.	Human Nail Bed	Flexible liquid-filled PDMS microchannel-based shear force skin sensor.	Up to 5 N of shear forces.	0.088 N^-1 of applied normal force.	–	Allows stretching of artificial skin similar to natural skin, allowing accurate shear force measurements. The intrinsically flexible nature of the sensor makes it conformable to curved surfaces and suitable for repeated shear force measurement without significant fatigue.	Requires improvements in its sensitivity and failure analysis. Cycle strain tests needed to determine robustness against repeated large strains.

Figure 7. Diagrammatic illustration of a simple, multilayered paper-based microfluidic chip with a manually controllable flap for the noninvasive sample preparation and detection of clenbuterol from swine hair samples using SERS. Adapted with permission from Ref. [112].

Figure 8. Operational overview of a chelex-based microfabricated device for on-chip purification and amplification of DNA (deoxyribonucleic acid) from human hair in integrated flow-through polymerase chain reaction (PCR). Adapted from Ref. [107].

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Data availability statement

No data was used for the research described in the article.