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Microneedle-based bioassays

Disease diagnosis and therapeutic efficacy can be monitored via a number of established bioassays that sample body fluids to assess and monitor health conditions. Traditional bioassays generally include several steps and start with invasive body fluid extraction procedures. These steps are painful and often require specialized techniques and tailored equipment, as well as the supervision of professional medical personnel. Innovations in engineering alternative bioassays to address these shortcomings are thus desired. Microneedles (MNs) represent promising tools to sample body fluids, in view of their minimal invasiveness, painlessness, and uncomplicated implementation. Recent progress in microfabrication and materials engineering, including the development of hollow and solid MNs with uniquely optimized architectures and multi-functional materials, has positioned MN-based platforms as prime candidates for bioassay solutions. In this minireview, we summarize the studies of MN-based platforms for detection and diagnosis. We categorize the platforms based on three different mechanisms: MNs as body fluid reservoirs, MNs integrated with electrochemical assays, and MNs engineered with colorimetric analyses. A discussion of design principles for MN-based bioassay platforms is presented. We also discuss the challenges and opportunities associated with MN-based bioassays in future clinical applications.


Introduction
Bioassays employ quantitative or qualitative analytical methods to detect biologically relevant target molecules in a living body. They play an essential role in disease diagnosis and disease management, especially in the clinic, where diverse biomarkers, metabolites, and therapeutic drugs are readily analysed from patient-derived samples. 1,2 Body uids, such as sweat, saliva, interstitial skin uid (ISF), and blood, are some of the target samples that can be investigated. 3 Of these, sweat, saliva, and ISF are composed of aqueous uids rich in electrolytes and macromolecules, and contain an overall lower protein content. This latter property allows us to identify drugs or biomarkers in their free form, which is a much easier process, especially when compared to blood. 4 Depending on the nature of the various body uids, distinct sampling and detection strategies can be engineered. Blood sampling is a ubiquitous diagnostic approach with which a pathophysiological status is assessed. 5 However, this approach is inextricably associated with invasive techniques, including hypodermic needling or nger pricking. These processes are considered undesirable and painful by many patients and pose further limitations, such as an increased risk of both localized and systemic infection. Consequently, researchers have resorted to high accuracy but less invasive alternatives to blood sampling, by targeting the skin. The skin is the largest organ of the human body, providing an abundant source of ISF, a targetable body uid. Skin tissue undergoes constant exchange of substances with blood vessels, making ISF an alternative source for biomarker detection, albeit with a slight delay in the dynamics of analytes. 6 For instance, glucose level uctuations in ISF are generally delayed by 4-10 min compared to those in blood. 7 Furthermore, from a sampling perspective, sourcing ISF is a more complicated and time-consuming technique in the clinic, though several techniques have been developed for ISF sampling, including using suction blisters, 8,9 reverse iontophoresis, 10 and microdialysis. 11,12 Unlike blood, ISF can also be continuously monitored as it does not clot. These strategies require specialized equipment that need to be operated by trained medical professionals, making a convenient and minimally invasive bioassay technique that is capable of readily sampling target uids highly desired.
The skin's outer epidermis structure is the major barrier to extracting body uid albeit a so and exible organ. One of the skin's primary biological functions is to act as the body's physical barrier to the external world, as well as providing homeostatic regulation and sensory perception. [13][14][15] The skin's structure can be generally divided into three layers: the epidermis, dermis, and hypodermisfrom the outermost to the innermost layer. Within the epidermis, the lower portion is an avascular environment with keratinocytes and ISF perfusion. 16 Cells residing in the epidermis layer are nourished by the nutrients diffused from blood microcirculation within the dermis. The dermisor dermal layer, provides a major source for ISF sampling, and both ISF and blood in the dermis can be sampled for diagnostic applications. 17 In order to reach ISF-rich tissue, sampling techniques require the disruption of the epidermal barrier and its connective tissue with the dermis. Microneedles (MNs) are known to efficiently penetrate the skin barrier with minimal invasiveness and pain, and have been used in many biomedical elds ranging from transdermal drug delivery to biosensing. 18,19 Moreover, owing to their easy-to-use nature, MNs can pave the way to patient self-administering procedures, helping alleviate the demand for medical personnel and improve bioassay accessibility.
MN fabrication comprises a plethora of options, providing a versatile platform to engineer discrete geometries and architectures that can be specically tailored to the uid sampling requirement. For instance, MNs can be designed with a certain length, aspect ratio, and stiffness to pierce the stratum corneum (the outermost of the epidermis) and form microscale ISF extraction channels. Moreover, by tuning the geometric scale of MNs, they can be engineered to reach the blood-perfused dermis and enable direct blood extraction and detection. 20,21 Furthermore, MNs can be integrated with other detection strategies. In this minireview, we summarize the designs of MN platforms for bioassay use, including hollow MNs and solid MNs with different shapes and structures (Fig. 1A). Various strategies that have been utilized for MN fabrication are discussed (Fig. 1B). We also summarize detection and diagnosis mechanisms into three distinct MN platforms: MNs as body uid reservoirs, MN-based electrochemical assays, and MNs integrated with colorimetric analyses (Fig. 1C). Finally, we discuss the challenges and opportunities for future clinical applications of MN-based bioassay platforms.

Design and fabrication strategies of MNs
MN-based bioassays establish a connective interface between body uid and the MN matrix by a minimally invasive penetration of a dened area of the skin. The design and fabrication of MNs determine their specic bioassay features. Many strategies (e.g., etching, 22,23 lithography, 24-26 micromachining, [27][28][29] and threedimensional (3D) printing [30][31][32] ) have been used for these purposes. Etching is one of the traditional methods to directly fabricate MNs through chemical reagents or physical ions, and is especially used to produce metal-based MNs. 33 Jin et al. fabricated solid steel MN patches by laser micro-etching techniques, that were subsequently coated with reduced graphene oxide (rGO) and Pt nanohybrids. The MNs functioned as an electrochemical system for minimally invasive transdermal detection of H 2 O 2 . To avoid mechanical destruction of the Pt/rGO layer during the skin insertion process, the authors also engineered a watersoluble polymer (polyvinylpyrrolidone) that was assembled outside the nanohybrid layer. Real-time sensing of H 2 O 2 in living mice was achieved by using the MN electrodes, with the nanohybrid coating shown to signicantly improve detection sensitivity. 34 In spite of their ability to detect subdermal biomarkers in situ, solid metal MN-based electrodes were unable to extract body uid and thus presented limited use in multi-biomarker detection. Alternative designs are required to potentiate extraction capabilities for MNs, such as using hollow MNs. Through a deepreactive ion etching method, Li et al. fabricated silicon hollow MNs with the aim of monitoring biomarkers in ISF. The MNs presented circular holes with diameters of 30 mm, and a depth of >300 mm was achieved via the efficient elimination of uorocarbon passivation polymers by ion bombardment. 35 In contrast, advances in additive manufacturing, such as 3D printing, have provided an alternative strategy to design and fabricat MNs with bespoke structures. [36][37][38] Yeung et al. utilized stereolithographybased 3D printing to create hollow MNs interfaced with micro-uidic structures. In contrast to previous work showing only 3D printed MN devices, the authors integrated microuidics and MNs in one step. These 3D printed and microuidic-integrated hollow MNs can be adopted in future biomedical devices targeted at transdermal drug delivery and biouid extraction, the latter being an essential element in bioassays. 39 Aside from fabricating MNs directly, the most commonly used approach for MN fabrication is molding, which can realize accurate duplicates of designed structures and allow the use of various materials otherwise unsuitable for etching and 3D printing. [40][41][42] The designed master template can initially be produced via etching, lithography, micromachining, or 3D printing. 43,44 Complementary negative molds can be produced with curable materials such as polydimethylsiloxane (PDMS). Some negative molds are commercially available with customizable designs, which can enhance the productivity, reduce the batch-to-batch variability, and facilitate access to MN fabrication techniques. To improve aspect ratios and tip sharpness of MNs, Krieger et al. developed a simple MN mold-fabrication technique using a low-cost desktop stereolithography (SLA) 3D printer. The authors printed MNs and subsequently carried out silicone casting. The female silicone mold was obtained aer degassing and heating. MNs composed of carboxymethyl cellulose and polylactic acid (PLA) were successfully manufactured via this silicone mold. 45 Chang et al. used stainless-steel master molds of MNs to fabricate a negative PDMS template. Hyaluronic acid (HA)-based MNs were efficiently produced through this mold, with the swellable MNs shown to be capable of rapid extraction of skin ISF for timely metabolic analysis. 46 Overall, different fabrication strategies can enable the manufacture of MNs from different materials. Direct fabrication approaches (etching, lithography, micromachining, and 3D printing) are compatible with metals, silicon ceramics, and polymers. In contrast, the indirect approach based on molding is mostly for making polymer-based MNs.
Based on the aforementioned manufacturing techniques, diverse raw materials (such as metals, 47,48 silicon, 49,50 ceramics, 51,52 and polymers [53][54][55] ) have been utilized to fabricate MN patches containing arrays of solid, 56,57 porous, 58,59 or hollow 60,61 needles. Samant et al. investigated the effects of the size of MNs on pressure-driven extraction of ISF, and the results showed a decreased volume of collected ISF with the increase of MN length between 250 mm and 750 mm. However, these effects were shown to be negligible (no statistically signicant difference). 62 To improve the extraction and detection efficiencies, MNs with novel microstructures or materials can be prepared by a multi-step process. [63][64][65][66][67] For example, Mandal et al. designed a MN patch with a core-shell structure for longitudinal sampling of cells and skin ISF. The group rst fabricated polylactic acid (PLA) MN arrays through PDMS molds and subsequently coated them with polylysine, alginate, and sucrose mixtures. By incorporating molecular adjuvants and specic antigen nanocapsules into the alginate hydrogel coating, antigen-specic lymphocytes could be enriched in the extracted cell populations, allowing for subsequent phenotypic and functional analysis. 68 Moreover, Sulaiman et al. coated an alginate-peptide nucleic acid hybrid material into the PLA MN arrays to obtain sequence-specic sampling, isolation, and detection of nucleic acid-based biomarkers from dermal ISF. 69 This core-shell MN-based technology was efficient as a diagnostic platform for biomarker extraction through hydrogel swelling and achieved the monitoring of patient-specic disease progression. Zhang et al. engineered photonic crystal (PhC) barcode-integrated MNs to enable multiplexed detection of dermal ISF biomarkers (Fig. 2). The biomarkers could be read out by uorescence intensity and the species of biomarkers were distinguished through the reection peaks of PhC barcodes. Compared to existing MNs used for ISF analysis, the advantages of these encoded MNs are their equivalent detection efficacies, largely simplied operational process, and simultaneous detection of multiple biomarkers. 70

Detection and diagnosis using MNs
Transdermal monitoring and detection of biomarkers using the MN platform are highly desirable for personalized diagnostics as well as point-of-care testing. 71 MNs have the potential to meet the need of different detection strategies, including being used   polio-specic neutralizing antibodies, and anti-polio IgG content in ISF was similar to that from serum. 80 The applications of MNs have also been expanded to blood testing, which is believed to be a more reliable body uid source for diagnostic purposes. Blicharz et al. developed a MN-based device for a onestep painless collection of capillary blood samples. As shown in Fig. 4, the authors integrated an array of solid MNs, a highvelocity MN insertion mechanism, stored vacuum, and a microuidic system containing a lithium heparin anticoagulant into a single compact device. Around 100 mL of blood samples could be collected each time by the single push of a button. The use of the device was shown to be simple and  This journal is © The Royal Society of Chemistry 2020 Nanoscale Adv., 2020, 2, 4295-4304 | 4299 straightforward, making it suitable for self-collection of blood samples at home. 81 When the MN platform serves as a body uid reservoir, a recovery step is normally necessary, which results in the need for further off-line analysis using laboratory equipment. To enable real-time analysis of body uid during extraction, MNs ought to be integrated with electrochemical sensing technology. 82,83 Electrodes can be inserted into MNs or MNs themselves can be engineered as electrodes by fabricating them with electroconductive materials. The detecting agents such as enzymes and chemical catalysts can be immobilized on MNs via coating, blending, or covalent bonding. A detection-relevant redox reaction can be initiated aer pushing the engineered MNs into the skin, where they interact with the biomarkers in the target uid. The sustained chemical reaction can be used to trigger an electrical signal, which can be interpreted by a detection instrument. 84-86 Wang's group developed an attractive skin-worn MN sensing device for the continuous detection of organophosphate (OP) nerve agents (Fig. 5). The sensor relied on the coupling of the biocatalytic action of organophosphorus hydrolase (OPH) with hollow pyramidal MNs. The MNs were modied with carbon paste array electrode transducers, which allowed the rapid voltammetric measurement of p-nitrophenol (a product from the OPH enzymatic reaction). The MN device presented a highly linear response for methyl paraoxon in the 20-180 mM range, with high selectivity and stability when exposed to ISF. 87 Given the pressing need to distinguish opioid overdoses from nerve agent poisoning, wearable MN devices can be engineered to provide a solution. A smart MN patch developed by Mishra et al. was the rst sensor to accomplish continuous monitoring of fentanyl concentration down to the nanomolar level through a nanomaterial-based multi-layered architecture. 88 Wang's group also designed hollow MNs integrated with Pt and Ag wires for interference-free ethanol detection in articial ISF, which showed good sensitivity, stability, and response rates. In their case, the MN aperture was modied by electropolymerizing o-phenylene diamine onto the Pt wire micro-transducer, followed by the immobilization of alcohol oxidase in an intermediate chitosan layer along with an outer Naon layer. 89 To improve the capture efficiency for tumor-related biomarkers, Yang et al. designed a wearable platform that can capture cell-free DNA from ISF within 10 min of application, by exploiting a dual-extraction effect created by reverse iontophoresis and MNs. 90 Another application of electrochemistry-based MN devices is continuous glucose monitoring, which could greatly help improve diabetes management. 91,92 Bollella et al. developed a porous gold MNbased biosensor for minimally invasive monitoring of glucose. The MN electrode was prepared by gold electrodeposition, and the porous gold surface was immobilized with 6-(ferrocenyl) hexanethiol (a redox mediator) and avin adenine dinucleotide glucose dehydrogenase. The designed MN-based biosensor showed remarkable performance for glucose detection in arti-cial ISF with an extended linear range, high sensitivity, stability, selectivity, and short response time. 93 Kim et al. fabricated MN electrodes based on glucose oxidase, and the integrated device was made wireless to allow remote glucose monitoring via a Bluetooth-enabled mobile phone. 94 Since enzyme-based MN devices have demonstrated instability in systems with unsuitable temperature or pH (due enzymatic denaturing), non-enzymatic MN sensors can provide signicant advantages in glucose detection. 95 Chinnadayyala et al. fabricated a stainless-steel MN patch by coating the tips with gold, Naon, and platinum black for non-enzymatic (direct) sensing  of glucose. The sensor presented good reproducibility and stability even in an acidic environment. 96 In addition, electrochemical technology-based MN devices also showed various applications in ion monitoring (e.g., lithium, 97 potassium 98 ) and metabolite detection (e.g., lactate, 99 urea 100 ) in vivo.
Recently, MN platforms for bio-uid extraction and biomarker detection via colorimetric reactions attracted signicant interest, especially as they bypass the need for complex in-lab apparatus, and pave the way for future use in self-diagnosis at home. [101][102][103] He et al. developed a hydrogel MN patch made of polyvinyl alcohol (PVA) and chitosan (CS) for point-of-care testing. Aer the extraction process, the target biomarkers from the MN patch were rapidly recovered by gentle heating because the PVA/CS hydrogel is water-soluble at higher temperatures. The recovered biomarkers (glucose, chlorine, lactate, or BSA) were successfully detected using a colorimetric method without the need for any bulky instrument. 104 Furthermore, to simplify the detection process and improve the operability of the device for end-users, an all-in-one uid sampling and result displaying transdermal colorimetric MN patch was developed. Wang et al. designed a double-layered MN patch with dual enzyme systems for simultaneous glucose sensing and color conversion. The coloration of 3,3 0 ,5,5 0 -tetramethylbenzidine was triggered by the cascade enzymatic reactions of glucose oxidase and horseradish peroxidase at high glucose levels in dermal ISF. The color could be visualized by the naked eyes and further quantied by imaging (Fig. 6). 105 Li et al. developed an all-in-one multi-diagnostic system for blood collection, serum separation, and biomarker detection. They integrated a hollow MN with a paper-based sensor to detect glucose and cholesterol in rabbits. 106 Besides the aforementioned approaches, novel detection methods are emerging. A new MN platform was integrated with surface-enhanced Raman spectroscopy (SERS) for sensing chemicals in ISF. The polymeric MNs were coated with gold nanorods or silver. With the immobilization of different functional molecules (4-mercaptobenzoic acid or 1-decanethiol) in the coating layer, the platform was capable of measuring pH 65 or quantifying glucose. 67 In another study, Yuan et al. designed a theranostic MN probe combining optical coherence tomography imaging with laser ablation. The MN achieved real-time imaging as well as ablation of tumors deep in a mouse brain. 107

Summary and outlook
MN-based bioassays are minimally invasive strategies compared to traditional sampling strategies that generally involve syringes and needles. This feature could potentially relieve the burden on medical resources, improve the efficiency of healthcare services, as well as improve patient compliance. In addition, integrative or all-in-one designs, can be achieved in MN platforms, which could further improve the user-friendly feature of these solutions, especially if test accuracy is maintained and user-dependent variability is controlled. Here, we reviewed the design and fabrication strategies of MNs with a focus on their applications for detection and diagnosis. The MN-based bioassays covered in this review are summarized in Table 1. As increasing research efforts have been devoted to MN-based body uid extraction and bioassays, there is an emerging trend for developing better MNbased real-time assays for personalized or point-of-care devices. However, challenges remain for MN-based bioassays, and additional design strategies ought to be considered. For instance, adhesiveness could be added to current designs since sufficient extraction of body uids requires extended time. Unstable physical adherence of MN patches may cause the MN tips to shed off and decrease the extraction efficiency. Many studies pertaining to the use of MNs in drug delivery demonstrate that MNs with barbs are able to prevent their tips from falling off the skin; 108 however, this structure may not be suitable for MN-based extraction and detection since the locked MN tips may be hard to remove aer body uid extraction. Indeed, the balance between xation and separation of MNs during body uid extraction is critical. Furthermore, continuous efforts are needed for material innovations to improve liquid absorption capabilities of MNs. The development of MNs capable of capturing and detecting molecules from body uids (such as antibodies, metabolites, and drug intermediates) will be an important step forward. In addition, most of the MN-based bioassay studies are tested on small animals (mice, rats, and rabbits). In order to translate their application for human use, the scale and design of MNs may need to be tailored to t the physiological features of the human skin. A novel MN-based device capable of blood extraction has been approved by the US Food and Drug Administration (FDA). 81 It paves the way for future commercialization of MN-based bioassay platforms. With continuous progress made in the development of MN devices, MN-based bioassays will serve as a minimally invasive alternative to traditional detection methods in clinics and function as a crucial intermediate step for the future of personalized healthcare.

Conflicts of interest
There are no conicts to declare.