Microdroplet Array Research Articles

Droplet array with microfluidic concentration gradient (DA-MCG) for 2-dimensional reaction condition screening

Droplet microfluidic technology can use each microdroplet as a microreactor, which has the advantages of low reagent dosage, less cross contamination, and fast reaction time. Combining concentration gradient generation with droplet formation and capture to form a two-dimensional reaction condition screening platform (including reactant concentration and reaction time) is expected to broaden the application range of microfluidic screening. In this work, a microfluidic chip that can dynamically generate and capture microdroplets and form static microdroplet array was designed and fabricated. An optimized Christmas tree structure by adjusting the horizontal channel length ratio was used to generate a chemical concentration gradient while obtaining a uniform outlet flow rate, forming a droplet array with different concentrations. The performance of droplet array with microfluidic concentration gradient (DA-MCG) was verified using sodium fluorescein as a model reagent. The chromogenic reaction of NaOH and phenolphthalein, and luminol chemiluminescence reaction were used to verify the two-dimensional screening ability of DA-MCG. The results indicated that the DA-MCG has the potential to be applied in the field of multi-dimensional drug screening.

Facile and Rapid Microcontact Printing of Additive-Free Polydimethylsiloxane for Biological Patterning Diversity.

The development and application of micropatterning technology play a promising role in the manipulation of biological substances and the exploration of life sciences at the microscale. However, the universally adaptable micropatterning method with user-friendly properties for acceptance in routine laboratories remains scarce. Herein, a green, facile, and rapid microcontact printing method is reported for upgrading popularization and diversification of biological patterning. The three-step printing can achieve high simplicity and fidelity of additive-free polydimethylsiloxane (PDMS) micropatterning and chip fabrication within 8 min as well as keep their high stability and diversity. A detailed experimental report is provided to support the advanced microcontact printing method. Furthermore, the applications of easy-to-operate PDMS-patterned chips are extensively validated to complete microdroplet array assembly with spatial control, cell pattern formation with high efficiency and geometry customization, and microtissue assembly and biomimetic tumor construction on a large scale. This straightforward method promotes diverse micropatternings with minimal time, effort, and expertise and maximal biocompatibility, which might broaden its applications in interdisciplinary scientific communities. This work also offers an insight into the establishment of popularized and market-oriented microtools for biomedical purposes such as biosensing, organs on a chip, cancer research, and bioscreening.

Microarray Platforms Based on 3D Printing.

This paper introduces an innovative method for the fabrication and infusion of microwell arrays based on digital light processing (DLP) 3D printing. A low-cost DLP 3D printer is employed to fabricate microstructures rapidly with a broad dynamic range while maintaining high precision and fidelity. We constructed microwell arrays with varying diameters, from 200 to 2000 μm and multiple aspect ratios, in addition to microchannels with widths ranging from 45 to 1000 μm, proving the potential and flexibility of this fabrication method. The superimposition of parallel microchannels onto the microwell array, facilitated by positive or negative pressure, enabled the transfer of liquid to the microwells. Upon removal of the microchannel chip, a dispensed microdroplet array was obtained. This array can be modulated by adjusting the volume of the microwells and the inflow fluid. The filled microwell array allows chip-to-chip dispensing to the microreactor array through binding and centrifugation, facilitating multistep and multireagent assays. The 3D printing approach also enables the fabrication of intricate cavity designs, such as micropyramid arrays, which can be integrated with parallel microchannels to generate spheroid flowcells. This device demonstrated the ability to generate spheroids and manipulate their environment. We have successfully utilized precise modulation of spheroids size and performed parallel drug dose-response assays to evaluate its effectiveness. Furthermore, we managed to execute dynamic drug combinations based on a compact spheroids array, utilizing two orthogonal parallel microchannels. Our findings suggest that both the combination and temporal sequence of drug administration have a significant impact on therapeutic outcomes.

Dielectric Liquid Microlens Array with Tunable Focal Length Based on Microdroplet Array Created via Dip-Coating Method.

A dielectric liquid microlens array (LMA) with a tunable focal length was fabricated by using a microdroplet array generated through the dip-coating method. The process began with treating the octadecyltrichlorosilane (OTS) layer with selective UV/O3 irradiation for 20 min to establish a hydrophilic-hydrophobic patterning surface. The substrate was subsequently immersed in glycerol and then withdrawn at a constant rate to create a microdroplet array. Upon filling the cell with matching oil (SL5267) and placing it within a square array of a 200 μm diameter glycerol microdroplet array, the LMA was produced. The focal length ranged from approximately -0.96 to -0.3 mm within a voltage range of 0 to 60 Vrms. The glycerol microdroplets, characterized by their shapes, sizes, curvatures, and filling factors, can be precisely controlled by designing an OTS patterning or adjusting the dip-coating speed. This approach offers a rapid and high-throughput method for preparation. Our approach to fabricating tunable LMA offers several advantages, including simplicity of fabrication, uniform structural properties, cost-effectiveness, polarization independence, and excellent optical performance. These focus-tunable LMAs hold significant potential for applications in image processing, 3D displays, medical endoscopy, and military technologies.

Enhancing microdroplet array generation via heterogeneous inner surface Modification: Biomimetic & hydrophobic approach

Microdroplet array chips have great potential for biological applications. Both physical structure and chemical material modification are effective surface modification techniques for the generation of droplet arrays. However, single surface modification technique is constrained by either unstable infiltration state transition or the requirement for high surface tension. In this paper, we proposed a heterogeneous inner surface modification technique for an enclosed microdroplet array chip by coupling the physically structured surface modification that accelerates the wetting state of the aqueous solution, and the material-modified surface that can maintain this state, thus keeping the aqueous solution in a stable Wenzel state. This technique provides a stable surface energy difference for enhanced microdroplet array generation. We investigated the influences of variables that affect the heterogeneity of the inner surfaces of the chip, such as the contact angles of the upper and lower surfaces and the size of the micropores on the stability and size of the generated droplet. We also evaluated the compatibility of technique for different types of liquids and biological reagents for the droplet array generation. Furthermore, we demonstrated the potential of the droplet arrays generated in a heterogeneous dual-regulation manner for DNA amplification in loop-mediated isothermal amplification (LAMP).

Ultrafast Solid-State Electrochemical Imprinting Utilizing Polymer Electrolyte Membrane Stamps for Static/Dynamic Structural Coloration and Letter Encryption.

Micro and nanopatterned surfaces hold potential for various applications, such as wettability control, antibiofouling, and optical components. However, conventional patterning processes are characterized by complexity, high costs, and environmental burdens because of the use of resists. Therefore, this paper proposes facile and ultrafast electrochemical imprinting employing a polymer electrolyte membrane (PEM) stamp for achieving micro and nanoscale patterning on Si surfaces. The solid-state electrochemical process efficiently generates oxide and hydrated oxide (Si-OH) patterns within several seconds at room temperature in a dry ambient environment. The formed oxide pattern can be employed as an etching mask to prepare diffraction gratings with diverse high-resolution (≈100nm) patterns utilizing the dry PEM stamp. The resulting oxide pattern on the Si surface exhibits instantaneous structural coloration upon exposure to humid air, attributable to the formation of a water microdroplet array on the oxide pattern. The oxide pattern is successfully applied for dynamic diffraction grating and letter encryption. The proposed solid-state electrochemical oxidation scheme based on a PEM stamp, which eliminates the need for liquid electrolyte and resist, represents a simple and ultrafast process with a time cost of a few seconds, characterized by low processing costs and environmental impact.

Numerical investigation on cooperative evaporation from microdroplet array on heated substrate

The evaporative heat and mass transport characteristics from an array of continuously fed microdroplet on a heated substrate are investigated numerically by a Multiphysics model, which incorporates heat conduction, buoyant flow, Marangoni flow, Stefan flow, and vapor diffusion. The effects of droplet spacing, contact angle, and droplet size on the evaporation rate, heat flux, and convection strength were analyzed in detail with a fixed thermal and vapor concentration boundary condition. The results revealed the existence of extremely strong convection current in the ambient gas domain for evaporation from droplet array. This convection effect dominates the vapor transport process, overcomes the suppression effect from neighboring droplet, and causes the total evaporation rate to exceed the prediction from traditional diffusion-based model by up to ten times. The strength of the convective vapor transport is characterized by a dimensionless parameter, which increases first from 2.3 to 7 and then decrease to 4 with increasing contact angle from 30° to 150°, but remains invariant at 6.3 for hemispherical droplet irrespective of the change in droplet dimension. Finally, the numerical results demonstrate potential for microdroplet array evaporation to resolve the thermal management challenge of ultrahigh power electronics with heat flux up to 1 kW/cm2.

Enhancement of solute diffusion in microdroplets using microrotors under rotational magnetic field

In vertical contact control (VCC), a microdroplet array selectively contacts with an opposite microdroplet array. Generally, VCC is useful for the dispenser mechanism based on solute diffusion between microdroplet pairs. However, sedimentation due to gravity can cause an inhomogeneous distribution of solutes in microdroplets. Therefore, it is necessary to enhance solute diffusion to achieve the accurate dispensing of a large quantity of solute in the direction opposite to that of gravity. Herein, we applied a rotational magnetic field to the microrotors in microdroplets to enhance the solute diffusion in microdroplets. Driven by microrotors, the rotational flow can generate a homogeneous distribution of solutes in microdroplets. We analyzed the diffusion dynamics of solutes using a phenomenological model, and the results showed that the rotation of microrotors can increase the diffusion constant of solutes.

Creating liquid crystal microdroplet arrays for multiplexed sensing by spatially-controlled molecular patterning

In this study, we presented a spatially-controlled molecular patterning technique to prepare liquid crystal (LC) microdroplet arrays on glass substrates. This technique utilizes an oxygen plasma activated PDMS stamp to remove the pre-coated dimethyloctadecyl 3-(trimethoxysilyl) propyl ammonium chloride (DMOAP) molecules from glass substrates, producing a surface with complementary patterns and specific hydrophobicity. When LC molecules are introduced onto this produced molecular pattern, a LC microdroplet array with uniform droplet size and positional order is formed. By using the LC microdroplet array doped with a Hg2+ selective ligand, the lowest detectable concentration of Hg2+ is 200-fold lower than that of conventional LC-based sensors. In addition, multiplexed detection of Hg2+, Al3+, Fe3+ and pH value in real water samples with a high recovery rate (∼100%) was demonstrated by using the LC microdroplet arrays doped with different molecular probes. Due to the specific arrangement of the LC microdroplet arrays, the collected optical signal of LC microarrays can be transformed into numerical symbols. The developed spatially controlled molecular patterning technique could be used to prepare highly uniform LC microdroplet arrays with consistent optical signals, providing a simple method to prepare the LC-based sensing platforms with improved sensing performance.

Programmable-Modulated Ultrasonic Transducer Array for Contactless Detection of Viral RNAs.

The current polymerase chain reactions-based nucleic acid tests for large-scale infectious disease diagnosis are always lab-dependent and generate large amounts of highly infectious plastic waste. Direct non-linear acoustic driven of microdroplets provide an ideal platform for contactless spatial and temporal manipulation of liquid samples. Here, a strategy to programmable-manipulate microdroplets using potential pressure well for contactless trace detection is conceptualized and designed. On such contactless modulation platform, up to seventy-two piezoelectric transducers are precisely self-focusing single-axis arranged and controlled, which can generate dynamic pressure nodes for effectively contact-free manipulating microdroplets without vessel contamination. In addition, the patterned microdroplet array can act as contactless microreactor and allow multiple trace samples (1-5µL) biochemical analysis, and the ultrasonic vortex can also accelerate non-equilibrium chemical reactions such as recombinase polymerase amplification (RPA). The results of fluorescence detection indicated that such programmable modulated microdroplet achieved contactless trace nucleic acid detection with a sensitivity of 0.21copyµL-1 in only 6-14min, which is 30.3-43.3% shorter than the conventional RPA approach. Such a programmable containerless microdroplet platform can be used for toxic, hazardous, or infectious samples sensing, opening up new avenues for developing future fully automated detection systems.

Integrated microdroplet array platform with temperature controller and micro-stirring for ultra-fast SARS-CoV-2 detection

The outbreak of COVID-19 has created a huge challenge to global health systems. Experience in fighting the epidemic shows that the development of a rapid and sensitive POCT diagnostic platform for SARS-CoV-2 that can be deployed in situ is crucial to contain the outbreak. Here, we have developed a portable microdroplet detection platform that integrated temperature controller and micro-stirring for high-throughput and ultrafast COVID-19 diagnosis. Such a device uses a p-n junction (PN junction) as the temperature controller to adjust the temperature in a single microdroplet independently and precisely, ensuring the amplification of reverse transcription loop-mediated isothermal amplification (RT-LAMP). Meanwhile, the platform incorporates an ultrasonic micro-stirring unit, greatly increasing the interaction between RT-LAMP molecules and accelerating the amplification. The results show good linearity over a wide linear range (1 to 105 copies/μL) and low LOD (0.48 copy/μL). Our method reports in only 6.1 min for high-viral load samples, and combines with sample preparation, the total detection process could be done within 30 min. Such a portable and fully integrated microdroplet molecular diagnostic platform is a promising tool for point-of-care diagnosis of COVID-19 and other infectious diseases in resource-limited settings.

Electrochemical immunosensor based on superwettable microdroplet array for detecting multiple Alzheimer's disease biomarkers.

Alzheimer's disease (AD) is a neurodegenerative disease caused by neurons damage in the brain, and it poses a serious threat to human life and health. No efficient treatment is available, but early diagnosis, discovery, and intervention are still crucial, effective strategies. In this study, an electrochemical sensing platform based on a superwettable microdroplet array was developed to detect multiple AD biomarkers containing Aβ40, Aβ42, T-tau, and P-tau181 of blood. The platform integrated a superwettable substrate based on nanoAu-modified vertical graphene (VG@Au) into a working electrode, which was mainly used for droplet sample anchoring and electrochemical signal generation. In addition, an electrochemical micro-workstation was used for signals conditioning. This superwettable electrochemical sensing platform showed high sensitivity and a low detection limit due to its excellent characteristics such as large specific surface, remarkable electrical conductivity, and good biocompatibility. The detection limit for Aβ40, Aβ42, T-tau, and P-tau181 were 0.064, 0.012, 0.039, and 0.041pg/ml, respectively. This study provides a promising method for the early diagnosis of AD.

Elucidating ejection regimes of metal microdroplets in voxel-based laser-induced forward transfer

Voxel-based laser-induced forward transfer (VB-LIFT) is a promising micro additive manufacturing process to efficiently print microstructures by ejecting a metal microdroplet array. Elucidating the ejection regimes of microdroplets is crucial to manipulating the printing process. This study investigates the ejecting behaviors of microdroplets induced by irradiating a femtoliter metal voxel with an ultraviolet nanosecond laser. A time-resolved dark-field imaging system was developed to capture the ejecting microdroplets. The ejecting velocity increased linearly with the laser fluence but was independent of the voxel side length. The analysis of the laser energy conversion revealed that one-thousandth of the laser energy was converted to the kinetic energy of the ejecting microdroplets, and the majority of the remaining were converted into internal energy. Meanwhile, the relaxation of the thermal compressive stress induced by laser heating drove the metal voxel detaching from the donor glass to generate a microdroplet. Furtherly, the ejection map in the VB-LIFT is proposed, including the non-detaching, detaching without melting, detaching and melting, and broken regimes. This study provides insights to eject debris-free metal microdroplets with the desired temperature and velocity in laser-induced forward transfer. • We develop a dark-field imaging system to capture the ejecting metal microdroplets. • Ejecting velocity is proportional to laser fluence but independent of voxel length. • Metal voxel detaching is driven by thermal compressive stress due to laser heating. • An ejection map containing four regimes is proposed for the VB-LIFT process.

Discontinuously Dewetting Solvent Arrays: Droplet Formation and Poly-Synchronous Surface Extraction for Mass Spectrometry Imaging Applications.

We describe a new liquid tissue stamping method called poly-synchronous surface extraction (PSSE) that utilizes an omniphobic substrate patterned with hydrophilic surface energy traps (SETs), which when wet with a solvent form a dense microdroplet array. When contacted with a tissue sample, each droplet locally extracts analytes from the tissue surface, which subsequentially can be analyzed by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-IMS) or ambient ionization-MS techniques. Optimization of the patterned surface with six different solvents was carried out to increase the droplet density, height, and reproducibility of volume deposition. Once optimized, sister slices of a strawberry (Fragaria × ananassa) were spatially extracted using the PSSE technique and the chemical distribution of selected compounds was analyzed with both MALDI-IMS and a lower resolution but faster ambient liquid microjunction surface sampling probe (LMJ-SSP) approach. Heat maps for target analytes for the PSSE approach are compared to those produced using traditional MALDI-IMS analysis. The PSSE method aligned well with direct analysis and demonstrated the potential to increase the speed of ambient MS tissue imaging techniques by decreasing the number of steps required for sample preparation.

Enhanced Isothermal Amplification for Ultrafast Sensing of SARS-CoV-2 in Microdroplets.

Rapid and high-throughput screening is critical to control the COVID-19 pandemic. Recombinase polymerase amplification (RPA) with highly accessible and sensitive nucleic acid amplification has been widely used for point-of-care infection diagnosis. Here, we report an integrated microdroplet array platform composed of an ultrasonic unit and minipillar array to enhance the RPA for ultrafast, high-sensitivity, and high-throughput detection of SARS-CoV-2. On such a platform, the independent microvolume reactions on individual minipillars greatly decrease the consumption of reagents. The microstreaming driven by ultrasound creates on-demand contactless microagitation in the microdroplets and promotes the interaction between RPA components, thus greatly accelerating the amplification. In the presence of microstreaming, the detection time is 6-12 min, which is 38.8-59.3% shorter than that of controls without microstreaming, and the end-point fluorescence intensity also increased 1.3-1.7 times. Furthermore, the microagitation-enhanced RPA also exhibits a lower detection limit (0.42 copy/μL) for SARS-CoV-2 in comparison to the controls. This integrated microdroplet array detection platform is expected to meet the needs for high-throughput nucleic acid testing (NAT) to improve the containment of viral transmission during the epidemic, as well as provide a potential platform for the timely detection of other pathogens or viruses.

Fish Capsules: A System for High‐Throughput Screening of Combinatorial Drugs (Adv. Sci. 9/2022)

High-Throughput Screening of Combinatorial Drugs In article number 2104449, Peng Shi, Honglin Li, Xudong Lin, and co-workers develop a novel fish capsules (FC) system, that is based on an automated zebrafish encapsulating technology and a microdroplet array strategy, to enable large-scale immobilization of oriented zebrafish larvae and compound parallel addition. Coupling with bioinformatic analysis and machine learning algorithms, this system is successfully demonstrated for in vivo high-throughput functional screening of mono/poly-therapies.

Wireless USB-like electrochemical platform for individual electrochemical sensing in microdroplets

The portable electrochemical platform can adapt to more usage scenarios, which is essential for drug discovery, toxicology, and cell biology. Here, we constructed the wireless USB-like electrochemical platform for simultaneous multi-channel electrochemical sensing in microdroplet array. On such platform, multi-channel mini-pillar arrays can anchor reagent droplet (a few microliters to dozens of microliters), and the wireless USB-like electrochemical platform can achieve simultaneously multi-channel detection. As a concept-of-proof for multi-channel, the 4∗4 channels are chosen as a model to proof the practicality of such portable electrochemical platform, the relative standard deviation (RSD) of such a platform in microdroplets sensing is below 5% level. The quantitative electrochemical analysis of multiple glucose concentrations in miniature workstation was also achieved, and only 0.97% difference between traditional electrochemical workstation. The wireless USB-like electrochemical platform with the characteristics of cloud data management and multi-channel, which enables remote detection and analysis and presents an opportunity for future portable high-throughput biomedical applications.

Photolithographic Patterning of FluorAcryl for Biphilic Microwell-Based Digital Bioassays and Selection of Bacteria.

FluorAcryl 3298 (FA) is a UV-curable fluoroacrylate polymer commonly employed as a chemically resistant, hydrophobic, and oleophobic coating. Here, FA was used in a cleanroom-based microstructuring process to fabricate hydrophilic-in-hydrophobic (HiH) micropatterned surfaces containing femtoliter-sized well arrays. A short protocol involving direct UV photopatterning, an etching step, and final recovery of the hydrophobic properties of the polymer produced patterned substrates with micrometer resolution. Specifically, HiH microwell arrays were obtained with a well diameter of 10 μm and various well depths ranging from 300 nm to 1 μm with high reproducibility. The 300 nm deep microdroplet array (MDA) substrates were used for digital immunoassays, which presented a limit of detection in the attomolar range. This demonstrated the chemical functionality of the hydrophilic and hydrophobic surfaces. Furthermore, the 1 μm deep wells could efficiently capture particles such as bacteria, whereas the 300 nm deep substrates or other types of flat HiH molecular monolayers could not. Capturing a mixture of bacteria expressing red- and green-fluorescent proteins, respectively, served as a model for screening and selection of specific phenotypes using FA-MDAs. Here, green-fluorescent bacteria were specifically selected by overlaying a solution of gelatin methacryloyl (GelMA) mixed with a photoinitiator and using a high-magnification objective, together with custom pinholes, in a common fluorescence microscope to cross-link the hydrogel around the bacteria of interest. In conclusion, due to the straightforward processing, versatility, and low-price, FA is an advantageous alternative to more commonly used fluorinated materials, such as CYTOP or Teflon-AF, for the fabrication of HiH microwell arrays and other biphilic microstructures.

Transport of Oligopeptide from Aqueous Phase to Span 80 Reverse Micelles in Microdroplet Array.

The partitioning of water and tetramethylrhodamine-conjugated-10-residue oligopeptides from the aqueous phase of microdroplets into Span 80 reverse micelles was observed by utilizing microdroplet arrays. Each peptide was dissolved in phosphate buffer saline, and initially encapsulated in arrayed droplets. An organic phase containing the reverse micelles was added to the microdroplets. Here, the hydration degree of the reverse micelle was adjusted by contact of the organic phase with a 1.0 M NaCl aqueous solution or with a phosphate buffer saline before combining it with the microdroplets. For micelles treated with a 1.0 M NaCl, significant water transport from the microdroplet to the micelle was observed, and peptide with low solubility in water was transported to the reverse micelles, while those with high solubility in water were not. For micelles treated with phosphate buffer saline, the water transport was minimal, and no significant peptide transport was observed. These results suggest that the partitioning of low-solubility oligopeptides requires accompanying water transport to the reverse micelle phase.

Directional anchoring patterned liquid-infused superamphiphobic surfaces for high-throughput droplet manipulation.

High-throughput experiments involving isolated droplets based on patterned superwettable surfaces are important for various applications related to biology, chemistry, and medicine, and they have attracted a large amount of interest. This paper provides a directional anchoring liquid-infused superamphiphobic surface (DAS), via combining concepts based on the droplet-anchoring behavior of beetle backs with patterned wettability, the directional adhesion of butterfly wings, and the slippery liquid-infused surfaces (SLISs) of pitcher plants. Regularly arranged ">"-shaped SLIS patterns were created on a superamphiphobic (SAM) background through ultrafast-laser-based technology. Improved directional anchoring abilities with a sliding angle difference of 77° were achieved; this is the largest sliding angle difference in a one-dimensional direction achieved using an artificial surface, to the best of the authors' knowledge. Thanks to the directional anchoring abilities, the DAS coupled droplet 'anchoring' and 'releasing' abilities. Furthermore, a high-throughput droplet manipulation device was designed, on which a micro-droplet array with a large number of droplets can be 'captured', 'transferred', or 'released' in a single step. With the addition of lubricant, the DAS can work continuously for even more than 30 cycles without cross-contamination between different droplets. The DAS also shows good stability under an ambient atmosphere and can maintain its functionality when manipulating corrosive droplets. The DAS and corresponding high-throughput droplet manipulation method are excellent candidates for practical applications.